Growth-promoting composition and its use

ABSTRACT

A combined formulation for IGF-I and growth hormone (GH) is useful for enhancing growth of a mammal. This formulation, which may be administered by infusion or injection to enhance growth, comprises IGF-I and GH, each in amounts of 0.1 to 100 mg/ml, in a pharmaceutically acceptable carrier at a pH of about 5-6 containing a surfactant, wherein the amounts of IGF-I and GH in the composition are effective to promote growth of a mammal more than an equivalent dose of IGF-I or GH alone, and wherein the weight ratio of IGF-I to GH in the composition ranges from 0.002:1 to 240:1.

This application is a continuation-in-part application of copending U.S.application Ser. No. 07/806,748 filed Dec. 13, 1991, now abandoned is adivisional application of U.S. application Ser. No. 07/535,005 filedJun. 7, 1990, now issued as U.S. Pat. No. 5,126,324.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to formulations containing IGF-I and growthhormone useful in a method of enhancing growth (anabolism) in patients,particularly those exhibiting a retarded growth rate or weight lossusing a combination of natural hormones.

2. Description of Related Art

Insulin-like growth factor I (IGF-I) is a polypeptide naturallyoccurring in human body fluids, for example, blood and human cerebralspinal fluid. Most tissues and especially the liver produce IGF-Itogether with specific IGF-binding proteins. These molecules are underthe control of growth hormone (GH). Like GH, IGF-I is a potent anabolicprotein. See Tanner et. al., Acta Endocrinol., 84: 681-696 (1977); Uthneet. al., J. Clin. Endocrinol. Metab., 39: 548-554 (1974)0. IGF-I hasbeen isolated from human serum and produced recombinantly. See, e.g., EP123,228 and 128,733.

Various biological activities of IGF-I have been identified. Researchershave found that an intravenous bolus injection of IGF-I lowers bloodglucose levels in humans, See Guler et. al., N. Engl. J. Med. 317:137-140 (1987). Additionally, IGF-I promotes growth in several metabolicconditions characterized by low IGF-I levels, such as hypophysectomizedrats [Guler et. al., Endocrinology, 118: Supp 129 abstract, Skottner et.al., J. Endocr., 112: 123-132 (1987); Guler et. al., Proc. Natl. Acad.Sci. USA, 85: 4889-4893 (1988); Froesch et. al., in Endocrinology, Intl.Congress Series 665, ed. by Labrie and Proulx (Amsterdam: ExcerptaMedica, 1984), p. 475-479], diabetic rats[Scheiwiller et. al., Nature,323: 169-171 (1986)], and dwarf rats [Skottner et. al., Endocrinology,124: 2519-2526 (1989)]. The kidney weight of hypophysectomized ratsincreases substantially upon prolonged infusions of IGF-Isubcutaneously. Guler et. al., Proceedings of the 1st European Congressof Endocrinology, 103: abstract 12-390 (Copenhagen, 1987). The kidneysof Snell dwarf mice and dwarf rats behaved similarly. van Buul-Offerset. al., Pediatr. Res., 20: 825-827 (1986); Skottner et. al.,Endocrinology, supra. An additional use for IGF-I is its administrationto improve glomerular filtration and renal plasma flow in humanpatients. See EP 327,503 published Aug. 9, 1989; Guler et. al., Proc.Natl. Acad. Sci. USA, 86: 2868-2872 (1989).

Human growth hormone (hGH) is a single-chain polypeptide consisting of191 amino acids (molecular weight 21,500). Disulfide bonds linkpositions 53 and 16.5 and positions 182 and 189. Nial., Nature, NewBiology, 230: 90 (1971). Human GH is a potent anabolic agent, especiallydue to retention of nitrogen, phosphorus, potassium, and calcium.Treatment of hypophysectomized rats with GH can restore at leastaportion of the growth rate of an intact animal. Moore et. al.,Endocrinology, 122: 2920-2926 (1988). Among its most striking effectsinhypopituitary (GH-deficient) subjects is accelerated linear growth ofbone growth plate cartilage resulting in increased stature. Kaplan,Growth Disorders in Children and Adolescents (Springfield, IL: CharlesC. Thomas, 1964).

In 1957, the mechanism of GH action was postulated as being due to GHinducing production of somatomedins (subsequently identified and namedIGF-I) in the liver, which travel via the circulation to produce all theeffects of GH. Salmon and Daughaday, J. Lab. Clin. Med., 49: 825-836(1957). Many studies investigating the relationships among GH, IGF-I,cartilage, cultured human fibroblasts, skeletal muscle, and growth havesupported this somatomedin hypothesis. See, e.g., Phillips andVassilopoulou-Sellin, N. Engl. J. Med., 302: 372-380; 438-446 (1980);Vetter et. al., J. Clin Invest., 7: 1903-1908 (1986); Cook et. al., J.Clin. Invest., 81: 206-212 (1988); Isgaard et. al., Endocrinology, 123:2605-2610 (1988); Schoenle et. al., Acta Endocrin., 108: 167-174 (1985).

Another theory holds that GH has a direct effect on chondrocytes that isnot dependent on circulating IGF-I. For example, several in vivo studieshave demonstrated longitudinal long bone growth in rats receiving hGHinjected directly into the tibial growth plate [Isaksson et. al.,Science, 216: 1237-1239 (1982); Russell and Spencer, Endocrinology, 116:2563-2567 (1985)] or the arterial supply to a limb [Schlechter et al.,Am. J. Physiol., 250: E231-235 (1986)]. Additionally it was found thatproliferation of cultured lapine ear and rib chondrocytes in culture isstimulated by hGH [Madsen et. al., Nature, 304: 545-547 (1983)], thisbeing consistent with a direct GH effect or with an indirect effect ofGH mediated by local GH-dependent IGF-I production. Such an autocrine orparacrine model for stimulation of growth has been supported by variouslines of experimental evidence. Schlechter et. al., Proc. Natl. Acad.Sci, USA, 83: 7932-7934 (1986); Nilsson et. al., Calcif. Tissue Int.,40: 91-96 (1987 ). Nilsson et. al. showed that while unilateral arterialinfusion of IGF-I did not produce a tibial longitudinal bone growthresponse in hypophysectomized rats, infusion of hGH did induce suchgrowth. Moreover, the influence of GH on the functional maturation ofhuman fetal islet cells in vitro could not be reproduced by addingIGF-I, suggesting a direct rather than a somatomedin-mediated action ofGH for these particular cells. Otonkoski et. al., Diabetes, 37:1678-1683 (1988).

A third theory for GH and IGF-I actions is that GH promotesdifferentiation of stem cells, rendering them responsive to stimulationof proliferation by IGF-I. Green et. al., Differentiation, 29: 195-198(1985). Although support for this model of GH acting to produce IGF-Ilocally, called the dual effector theory, has been obtained for certaincell types [Zezulak and Green, Science, 233: 551-553 (1986)], itsapplication to skeletal growth has not been established. It has beenfound that both GH and testosterone could stimulate skeletal growth inthe hypophysectomized prepubertal limb without alteration of circulatingIGF-I concentrations, the results not precluding the possibility thatthe growth-promoting effect of GH was affected by local actions at thesite of osteogenesis. Young et. al., J. Endocrin., 121: 563-570 (1989).Also, GH has been reported to stimulate tibial epiphyseal plate width inthe hypophysectomized rat without increasing circulating IGF-Iconcentrations. Orlowski and Chernausek, Endocrinol., 123: 44-49 (1988).

More recently, a study was undertaken to reproduce the "direct" in vitroGH effect on epiphyseal and articular chondrocytes to determine whetherthis effect is mediated by IGF-I in a local autocrine or paracrinefashion. =Trippel et. al., Pediatr. Res., 25: 76-82 (1989). Human GH wasfound not to stimulate rabbit articular or epiphyseal chondrocytes orbovine epiphyseal chondrocytes, whereas IGF-I stimulated both mitoticand differentiated cell functions in both epiphyseal and articularchondrocytes. The authors state that the data suggest that the role ofIGF-I in skeletal development is complex and may be diverse both in thecellular functions it regulates and the cell populations regulated,requiring further investigation to define the relationship of IGF-I toGH.

It has been reported that the growth response to co-addition of GH andIGF-I was not statistically different from that of GH alone when bodyweight gain, bone length, or tibial epiphyseal cartilage width wasmeasured. Skottner et. al., J. Endocr., supra [I.V. infusion of bGH (10mu/day) for 8 days and met-IGF-I (with specific activity of 3400 U/mg,120 μg/day) for the last 4 days]; Isgaard et. al., Am. J. Physiol., 250:E367-E372 (1986) [5 μg of IGF-I and 1 μg of hGH injected locally dailyfor 5 days]. It was also found that IGF-I, when injected or infusedsubcutaneously or infused intravenously, is a weak growth promoter inhypophysectomized rats compared with hGH, even when infused incombination with small amounts of hGH. Robinson and Clark, ActaPaediatr. Scand. supp., 347: 93-103 (1988).

As regards osteoblast-like cells in culture, direct stimulation of theirproliferation by hGH is at least partially mediated by IGF-I-likeimmunoreactivity [Ernst and Froesch, Biochem. Biophy. Res. commun., 151:142-147 (1988)]; the authors found that IGF-I and hGH had additiveeffects on osteoblast proliferation only when the exogenous IGF-Iconcentration exceeded that of endogenously produced IGF-I by a largemargin. Another in vitro study showed that purified human and syntheticIGF-I stimulated adult articular chondrocyte DNA and proteoglycansynthesis; GH had no effect on either process; and GH added incombination with IGF-I increased proteoglycan, cell-associatedproteoglycan, and keratan sulfatesynthesis over levels observed withIGF-I alone. Smith et. al., J. Orthop. Res., 7: 198-207 (1989). Separateadministration of hGH and IGF-I was found to enhance humangranulopoiesis, with the effect of hGH on marrow myeloid progenitorsapparently mediated by paracrine IGF-I. Merchav et. al., J. Clin.Invest., 83: 791-797 (1988). Merchav et. al. also noted that myeloidcolony formation was significantly enhanced in cultures stimulated withcombined limiting concentrations of both IGF-I and hGH, whereas combinedmaximal concentrations of both peptides did not exert an additiveeffect.

Also, based on recent immunohistochemical data regarding the GHreceptor, it has been suggested that GH may act independently of orsynergistically with therpatic IGF-I in carrying out itsgrowth-promoting role in the gastrointestinal tract. Lobie et. al.,Endorcrinol., 126: 299-306 (1990). It has been shown that pretreatmentof hypophysectomized rats with GH, but not with IGF-I, promotes theformation of chondrocyte colonies and makes the chondrocytes susceptibleto IGF-I in vitro. Lindahl et. al., Endocrinol., 121: 1070-1075 (1987).The authors suggest that GH induces colony formation byIGF-I-independent mechanisms and that IGF-I is a second effector in GHaction. Further, treatment of hypophysectomized animals with a singledose of hGH restored IGF-I mRNA in parenchyrnal and in non-parenchymalcells to the extent found in intact animals. van Neste et. al., J.Endocr., 119: 69-74 (1988).

However, it has also been reported that IGF-I directly suppresses GHgene transcription and GH secretion at the pituitary level in aninhibitory feedback control mechanism. Namba et. al., Endocrinol., 124:1794-1799 (1989); Yamashita et. al., J. Biol. Chem., 262:13254-13257(1987). Additionally, it was reported that the maximum stimulation ofglucose metabolism in 3T3 adipocytes achieved by hGH is only a fractionof that produced by various IGFs, indicating that extracellular IGFs donot mimic the effects of hGH on glucose metabolism in these adipocytes.Schwartz et. al., Proc. Natl. Acad. Sci. USA, 82: 8724-8728 (1985).Moreover, human GH was found not to enhance further the IGF,I-stimulatedLeydig cell steroidogenesis. Horikawa et. al., Eur. J. Pharmacol., 166:87-94 (1989). Another negative finding was that the combination of chickgrowth hormone and human IGF-I did not stimulate cell proliferation andmetabolic activity of cultured epiphyseal growth plate chondrocytesabove human IGF-I alone. Rosselot et. al., The Endocrine Society 72ndAnnual Meeting, abstract 202, p. 75, of Program and abstracts releasedprior to the meeting in Atlanta, GA on Jun. 20-23, 1990. It has alsobeen reported that both hGH and hGF-I can promote growth in the mutantdwarf rat, but they differ both quantitatively and qualitatively intheir pattern of actions. Skottner et. al., Endocrinology, supra.Additionally, a loss of IGF-I receptors in cultured bovine articularchondrocytes was found after pre-exposure of the cells topharmacological doses of either hGH or bGH. Watanabe et. al., J.Endocr., 107: 275-283 (1985). The necessity for large amounts of GH isattributed to extremely low affinity of GH binding sites on these cells.The authors speculate that living organisms have a protection mechanismto avoid unnecessary overgrowth of the body resulting in down-regulationof the IGF-I receptors.

U.S. Pat. No. 4,857,505 issued Aug. 15, 1989 discloses use of an adductof a growth hormone, growth factor, IGF-I, or fragment thereofcovalently bonded to an activated polysaccharide for increasedhalf-life, increased weight gain in animals, and increased milkproduction.

Known side effects of hGH treatment include sodium retention andexpansion of extracellular volume [Ikkos et. al., Acta Endocrinol(Copenhagen), 32: 341-361 (1959); Biglieri et. al., J. Clin. Endocrinol.Metab., 21: 361-370 (1961)], as well as hyperinsulinemia andhyperglycemia. The major apparent side effect of IGF-I is hypoglycemia.Guler et. al., Proc. Natl. Acad. Sci. USA, 1989, supra.

Various methods for formulating proteins or polypeptides have beendescribed. These include EP 267,015 published May 11, 1988; EP 308,238published Mar. 22, 1989; and EP 312,208 published Apr. 19, 1989, whichdisclose formulation of a polypeptide growth factor having mitogenicactivity, such as transforming growth factor-β (TGF-β), in apolysaccharide such as methylcellulose; EP 261,599 published Mar. 30,1988 disclosing human topical applications containing growth factorssuch as TGF-β; EP 193,917 published Sep. 10, 1986, which discloses aslow-release composition of a carbohydrate polymer such as a celluloseand a protein such as a growth factor; GB Pat. No. 2,160,528 grantedMar. 9, 1988, describing a formulation of a bioactive protein and apolysaccharide; and EP 193,372 published Sep. 3, 1986, disclosing anintranasally applicable powdery pharmaceutical composition containing anactive polypeptide, a quaternary ammonium compound, and a lower alkylether of cellulose. See also U.S. Pat. No. 4,609,640 issued Sep. 2, 1986disclosing a therapeutic agent and a water-soluble chelating agentselected from polysaccharides, celluloses, starches, dextroses,polypeptides, and synthetic polymers able to chelate Ca and Mg; and JP57/026625 published Feb. 12, 1982 disclosing a preparation of a proteinand water-soluble polymer such as soluble cellulose.

EP 123,304 published Oct. 31, 1984 discloses mixing tissue plasminogenactivator with gelatin or Polysorbate 80, and JP 58/224,687 publishedDec. 27, 1983 [Toryo, Chem. Abs., 100: 197765r (1984)] disclosedformulation of plasminogen-activating enzyme with PEG-3-sorbitanmonooleate, dextrin, gelatin, mannitol, dextran, glycine, and hydrolyzedgelatin for stability.

Furthermore, preservatives containing a quaternary ammonium salt havebeen added to chemical drug formulations to prevent growth of bacteria.See, e.g., Remington's Pharmaceutical Sciences, 18th edition (definitionof benzethonium chloride), Martindale, The Extra Pharmacopeia, 28thedition (p.550, entry on benzethonium chloride), United StatesPharmacopeia, 22nd edition (pp. 146-147, entries on benzethoniumchloride topical solution and tincture), Handbook on Injectable Drugs,5th edition (p. 246, entry on diphenhydrarnine HCl, which contains 0.1%benzethonium chloride; pp. 396-397, entry on ketamine HC1, whichcontains 0.1 mg/ml of benzethonium chloride; and pp. 695-696, entry onVidarabine, which contains 0.1 mg benzethonium chloride). Anotherexample is the formulation of octreotide in benzalkonium chloride fornasal application as described in GB Appln. 2,193,891 published Feb. 24,1988. The preservatives have been used in parenteral formulations at lowconcentrations, and in antiseptic washes for wound care at higherconcentrations. In addition, a mixture of a physiologically activepolypeptide with a quaternary ammonium compound and a lower alkyl etherof cellulose is disclosed, wherein the quaternary ammonium compound isadded to improve stability and preservability. EP 193,372.

It is an object of the present invention to provide a formulation usefulin promoting anabolic effects in mammals that combines GH and IGF-I.

It is another object to provide a formulation that has improved resultsover using IGF-I or GH alone in a formulation.

These and other objects will be apparent to those of ordinary skill inthe art.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a growth-promotingcomposition comprising IGF-I and GH, each in an amount of 0.1 to 100mg/ml, in a pharmaceutically acceptable carrier at pH about 6 containinga surfactant, wherein the amounts of IGF-I and GH in the composition areeffective to promote growth (anabolism) of a mammal more than anequivalent dose of IGF-I or GH alone, and wherein the weight ratio ofIGF-I to GH in the composition ranges from 0.002:1 to 240:1. Preferably,the amounts of IGF-I and GH in the composition are about 1 to 10 mg/mleach and the weight ratio of IGF-I to GH in the composition ranges from0.048:1 to 30:1. Also preferred is that the carrier be an acetic acidsalt buffer and a phosphate or citrate buffer, which carrier contains asodium counterion and a stabilizer. Additionally preferred is that thesurfactant is a polysorbate or poloxamer.

In a further aspect, the invention provides a composition comprisingIGF-I and GH in a weight ratio of IGF-I:GH of between about 2:1 and100:1 (w/w), about 0.05-0.3 mM of an osmolyte, about 0.1-10 mg/ml of astabilizer, about 1-5 mg/ml of a surfactant, and about 5-100 mM of abuffer at about pH 5-6.

In a still further aspect, the invention provides a method for enhancinggrowth of a mammal comprising administering to the mammal by eitherinjection or infusion an effective amount of the above composition so asto enhance the growth of the mammal over the enhancement in growthachieved using an equivalent dose of IGF-I or GH alone. Generally it wasfound that injection causes an overall growth in the man, but withoutconcurrently increasing the weight of the kidney or thymus, whereasinfusion causes not only an overall body weight increase, but alsoincreases the weights of the individual organs.

The literature shows that the role of IGF-I in skeletal development inconjunction with GH is complex, and evidence supporting various theoriesof GH action is contradictory and inconclusive. If GH acts viaproduction of circulating IGF-I (the somatomedin hypothesis), then amaximal dose of GH would not be expected to be enhanced by administeringIGF-I systemically. If GH acts locally to produce IGF-I, then it isunlikely that the high local concentrations of IGF-I predicted by thissecond theory could be reproduced by administering IGF-I eystemically.If some actions of GH do not involve IGF-I generation, then adding GHmight enhance the effect of IGF-I. However, in view of the confusionsurrounding which of these three unresolved theories is correct, therewas no clear basis to predict the outcome on body and bone growth ofadministering to a mammal a combination of GH and IGF-I.

Unexpectedly, a significantly greater daily body weight gain, increasedlongitudinal bone growth, and enhanced epiphyseal width of the tibiawere achieved after combination treatment with IGF-I and GH as comparedwith the same doses of each of IGF-I and GH alone. Further, the additiveeffect of IGF-I and GH was not seen for all tissues, indicating aselectivity for whole body growth, bone, and cartilage. Moreover, IGF-Ienhanced the growth-promoting effect of GH even at the maximum effectivedose of GH, and can further enhance a low dose of GH to produce amaximal growth response. Thus, IGF-I may be used in combination withlower doses of GH to increase growth of those immature patients thathave reached their maximum growth rate after treatment with maximaldoses of GH alone and then experienced a fall in their annualized growthrate. This is an effect that is widespread in all growth-deficientpatients after several months of treatment. The combination could alsobe used to maximize the growth response in patients who present late indevelopment with growth retardation, and only have a few years oftherapeutic intervention potential. Additionally, the combination can beused to treat those patients who exhibit side effects such asdiabetogenic symptoms with maximum doses of GH or hypoglycemia withmaximum doses of IGF-I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B represent graphs of cumulative body weight gain overseven days for each group of treated hypophysectomized adult male ratsfor two replicate studies 1 and 2, respectively, performed one monthapart (means±SD).

FIG. 2 shows a bar graph of the increase in width of epiphyseal bonegrowth plate after seven days of hGH and/or IGF-I treatment ofhypophysectomized rats (means±SD).

FIGS. 3A and 3B represent graphs of longitudinal bone growth andepiphyseal plate width (a separate study from FIG. 2), respectively, foreach group of hypophysectomized rats treated with hGH alone, or IGF-I ordes(1-3)-IGF-I alone or in combination with hGH (means±SD).

FIG. 4 illustrates a graph of weight gain in hypophysectomized rats overone week as a function of hGH concentration (log dose), where rats weretreated with IGF-I (2.4 mg/kg/day) using minipumps and with hGH dailyinjections (means SD).

FIG. 5 illustrates a graph of weight gain in dwarf rats over one week asa function of hGH concentration (log dose), where rats were treated withIGF-I (1.2 mg/kg/day) using minipumps and with hGH daily injections(means±SD).

FIG. 6 depicts a graph of weight gain in hypophysectomized rats usingthree different doses of IGF-I or des(1-3)-IGF-I infused subcutaneouslyby minipumps for seven days (means±SD).

FIG. 7 depicts a graph of weight gain in hypophysectomized rats usingthree different doses of hGH injected daily subcutaneously for sevendays (means±SD).

FIGS. 8A and 8B illustrate bar graphs of the growth rate in cm/year ofpatients of various growth inhibition etiologies having had either noprevious treatment (Prey Rx No) or previous treatment (Prev Rx Yes) withhGH. N indicates the number of patients at the indicated dose level ofhGH given in units of mg/kg. FIG. 8A is the data for the first year ofhGH treatment and FIG. 8B is for the second year of hGH treatment.

FIG. 9 illustrates bar graphs of the annualized (12-month) growth ratein cm/year of patients treated with the indicated dose of hGH in the1-2, 3-5, 6-8, 9-11, 12-14, 15-17, and more than 17 year ranges. Nindicates the number of patients in each age group.

FIG. 10 illustrates the percent change in blood glucose levels with timewhen either 7.50 (open) or 250 (filled) μg/rat of IGF-I is givensubcutaneously in two different formulations: pH 6.0 citrate (circles)and pH 5.0 acetate (triangles).

FIG. 11 illustrates the percent change in blood glucose levels with timewhen either 450 ] (open) or 150 (filled) μg/rat of IGF-I is givensubcutaneously in two different formulations: pH 6.0 citrate (circles)and pH 5.0 acetate (triangles).

FIG. 12 illustrates the percent change in blood glucose levels with timewhen 150 μg/rat of IGF-I in either the pH 6.0 citrate formulation (open)or pH 5.0 acetate formulation (filled) is given subcutaneously (circles)or intravenously (triangles).

FIG. 13 illustrates the percent change in blood glucose levels with timewhen either 450 (open) or 150 (filled) μg/rat of IGF-I is givensubcutaneously in two different formuations: pH 6.0 citrate (circles)and pH 5.0 citrate (triangles).

FIG. 14 illustrates the percent change in blood glucose levels with timewhen 150 μg/rat of IGF-I in either the pH 6.0 citrate formulation (open)or pH 5.4 acetate formulation (filled) is given subcutaneously (circles)or intravenously (triangles).

FIG. 15 shows the absolute glucose levels (mg %) when 150 μg/rat ofIGF-I is given in four different formulations: pH 6.0 citrate,administered subcutaneously (open circles), pH 5.4 acetate, administeredsubcutaneously (filled circles), pH 6.0 citrate, administered iv (opentriangles), and pH 5.4 acetate, administered iv (filled triangles).

FIG. 16 shows the percent change in blood glucose levels with time usingthe four different formulations, with the symbols being the same as forFIG. 15.

FIG. 17 shows the level of plasma IGF-I (ng/ml) versus time afterinjection of IGF-I (I50 μg/rat iv) using either the pH 6.0 citrateformulation (open circles) or pH 5.4 acetate formulation (filledcircles).

FIG. 18 is the same as FIG. 17 except that the IGF-I was administeredsubcutaneously instead of intravenously.

FIG. 19 shows the absolute glucose levels (mg %) when a pH 6.0 citrateIGF-I formulation (circles) was administered subcutaneously in doses of150 μg (open) and 450 μg (filled) and when a pH 5.4 acetate IGF-Iformulation (squares) was administered subcutaneously in doses of 50 μg(open) and 150 μg (filled).

FIG. 20 shows the percent change in blood glucose levels with time usingthe four different formulations, with the symbols being the same as forFIG. 19.

FIG. 21 shows the level of plasma IGF-I (ng/ml) versus time aftersubcutaneous injection of the four different formulations, with thesymbols being the same as for FIG. 19.

FIG. 22 shows the percent change in blood glucose levels with time innormal rats when a pH 6.0 citrate IGF-I formulation (triangles) wasadministered subcutaneously in doses of 900 μg (open) and 300 μg(filled) and when a pH 5.4 acetate IGF-I formulation (circles) wasadministered subcutaneously in doses of 300 μg (open) and 100 μg(filled).

FIG. 23 shows the percent change in blood glucose levels with time innormal rats when a pH 6.0 citrate IGF-I formulation (open circles) wasadministered subcutaneously in a dose of 300 μg, when a pH 5.4 acetateIGF-I formulation (filled circles) was administered subcutaneously in adose of 300 μg, and when a pH 5.4 acetate IGF-I and GH co-mixedformulation (triangles) was administered subcutaneously in doses of 100μg (open) and 10 μg (filled) of hGH and 300 μg IGF-I.

FIG. 24 shows the level of plasma IGF-I (ng/ml) versus time aftersubcutaneous injection of the four different formulations, with thesymbols being the same as for FIG. 23 for the co-mix, and with thefilled circles being the pH 6.0 IGF-I formulation and the filled squaresbeing the pH 5.4 IGF-I formulation alone.

FIG. 25 shows the body weight gain (g) in dw/dw rats administered bysubcutaneous injection excipient (open triangles), IGF-I at a dose of600 μg (filled triangles), rhGH at a dose of 30 μg (filled circles),rhGH at a dose of 120 μg (filled squares), IGF-I+hGH at doses of 600 μgand 30 μg IGF-I and hGH, respectively (open circles), and IGF-I+hGH atdoses of 600 μg and 120 μg IGF-I and hGH, respectively (open squares).

DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

As used herein, "mammal" signifies humans as well as animals, andincludes animals of economic importance such as bovine, ovine, andporcine animals. The preferred mammal herein is a human. The term"non-adult" refers to mammals that are from perinatal age (such aslow-birth-weight infants) up to the age of puberty, the latter beingthose that have not yet reached full growth potential.

As used herein, "IGF-I" refers to insulin-like growth factor from anyspecies, including bovine, ovine, porcine, equine, and preferably human,in native-sequence or in variant form, and from any source, whethernatural, synthetic, or recombinant. Preferred herein for animal use isthat form of IGF-I from the particular species being treated, such asporcine IGF-I to treat pigs, ovine IGF-I to treat sheep, bovine IGF-I totreat cattle, etc. Preferred herein for human use is humannative-sequence, mature IGF-I, more preferably without a N-terminalmethionine, prepared, e.g., by the process described in EP 230,869published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; or EP288,451 published Oct. 26, 1988. More preferably, this native-sequenceIGF-I is recombinantly produced and is available from Genentech, Inc.,South San Francisco, CA for clinical investigations. Also preferred foruse is IGF-I that has a specific activity greater than about 14,000units/mg as determined by radio receptor assay using placenta membranes,such as that available from KabiGen AB, Stockholm, Sweden.

The most preferred IGF-I variants are those described in PCT WO 87/01038published Feb. 26, 1987 and in PCT WO 89/05822 published Jun. 29, 1989,i.e., those wherein at least the glutamic acid residue is absent atposition 3 from the N-terminus of the mature molecule or those having adeletion of up to five amino acids at the N-terminus. The most preferredvariant has the first three amino acids from the N. terminus deleted(variously designated as brain IGF, tIGF-I, des(1-3)-IGF-I, ordes-IGF-I).

As used herein, "GH" refers to growth hormone from any species,including bovine, ovine, porcine, equine, and preferably human, innative-sequence or invariant form, and from any source, whether natural,synthetic, or recombinant. Preferred herein for animal use is that formof GH from the particular species being treated, such as porcine GHtotrear pigs, ovine GH to treat sheep, bovine GH to treat cattle, etc.Preferred herein for human use is human native-sequence, mature GH withor without a methionine at its N-terminus. Also preferred is recombinanthGH, i.e., that produced by means of recombinant DNA technology. Morepreferred is methionyl human growth hormone (met-hGH) produced in E.coli, by the process described in U.S. Pat. No. 4,755,465 issued Jul. 5,1988 and Goeddel et. al., Nature, 282: 544 (1979). Met-hGH, which issold under the trademark PROTROPIN® by Genentech, Inc., is identical tothe natural polypeptide, with the exception of the presence of anN-terminal methionine residue. This added amino acid is a result of thebacterial protein synthesis process.

Another preferred hGH for human use is a recombinant hGH (rhGH),available to clinical and research investigators from Genentech, Inc.under the trademark Nutropin®, and commercially available from EliLilly, that lacks this methionine residue and has an amino acid sequenceidentical to that of the natural hormone. See Gray et. al.,Biotechnology, 2: 161 (1984). Both meth-hGH and rhGH have equivalentpotencies and pharmacokinetic values. Moore et. al., supra.

As used herein, the term "growth" refers to the dynamics of staturalgrowth experienced by an individual during infancy, childhood, andadolescence as depicted by a normal growth curve. Thus, growth hereinrefers to the growth of linear-producing bone plate driven bychondrocytes, as distinguished from the growth of osteoblast cells,derived from a different part of the bone. Restoration of normal growthpatterns would allow the patient to approach a more satisfactory growthcurve. Examples of patients that are relatively resistant to GH butrequire treatment to induce an anabolic effect include those withTurner's Syndrome, GH deficient children who grow poorly in response toGH treatment, children who experience a slowing or retardation in theirnormal growth curve about 2-3 years before their growth plate closes, sothat GH administered alone would no longer increase growth of thechildren, so-called short normal children, and patients where the IGF-Iresponse to GH has been blocked chemically (i.e., by glucocorticoidtreatment) or by a natural condition such as in adult patients or incatabolic patients where the IGF-I response to GH is naturally reduced.

B. Modes for Carrying Out the Invention

The IGF-I and GH are directly administered to the mammal by any suitabletechnique, including parenterally, intranasally, intrapulmonarily, ororally. They need not be administered by the same route and can beadministered locally or systemically. The specific route ofadministration will depend, e.g., on the medical history of the patient,including any perceived or anticipated side or reduced anabolic effectsusing hGH or IGF-I alone, and the growth defect to be corrected.Examples of parenteral administration include subcutaneous,intramuscular, intravenous, intraarterial, and intraperitonealadministration. Most preferably, the administration is by continuousinfusion (using, e.g., minipumps such as osmotic pumps), or by injection(using, e.g., intravenous or subcutaneous means). Preferably, theadministration is subcutaneous for both IGF-I and GH. The administrationmay also be as a single bolus or by slow-release depot formulation.

Most preferably, the IGF-I is administered by injection, most preferablysubcutaneously, at a frequency of, most preferably, one-half, once,twice, or three times daily. Most preferably, the GH is administereddaily subcutaneously by injection. Co-injection of the IGF-I and GH isan optimal drug delivery system to ensure normal growth of the mammal,i.e., no overgrowth. Hence, delivery of hGH and IGF-I by injection willbe the preferred form of administration for body growth/anabolism, as itwill preserve normal body proportions.

In addition, the IGF-I is suitably administered together with itsbinding protein, for example, BP53, which is described in WO 9/09268published Oct. 5, 1989, which is equivalent to U.S. Pat. No. 5,258,280,and by Martin and Baxter, J. Biol. Chem., 261: 8754-8760 (1986), thedisclosures of which are incorporated herein by reference. Thisadministration may be by the method described in U.S. Pat. No.5,187,151. This protein is an acid-stable component of about 53 Kd on anon-reducing SDS-PAGE gel of a 125-150 Kd glycoprotein complex found inhuman plasma that carries most of the endogenous IGFs and is alsoregulated by GH. The IGF-I is also suitably coupled to a receptor orantibody or antibody fragment for administration. Similarly, the GH canbe delivered coupled to another agent such as an antibody, an antibodyfragment, or one of its binding proteins.

The IGF-I and GH composition(s) to be used in the therapy will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with hGH or IGF-I alone orgrowth retardation after continuous GH treatment), the site Of deliveryof the IGF-I and GH composition(s), the method of administration, thescheduling of administration, and other factors known to practitioners.The "effective amounts" of each component for purposes herein are thusdetermined by such considerations and must be amounts that enhancegrowth of the treated patient over growth enhancement that is obtainedusing the same amount of IGF-I or GH individually.

As a general proposition, the total pharmaceutically effective amount ofeach of the IGF-I and GH administered parenterally per dose will be inthe range of about 1 μg/kg/day to 100 mg/kg/day of patient body weight,although, as noted above, this will be subject to a great deal oftherapeutic discretion. More preferably, this dose is at least 0.1mg/kg/day, and most preferably at least 1 mg/kg/day for each hormone. Ifgiven continuously, the IGF-I and GH are each typically administered ata dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4injections per day or by continuous subcutaneous infusions, for example,using a minipump. An intravenous bag solution may also be employed. Thekey factor in selecting an appropriate dose is the result obtained, asmeasured by increases in body weight gain, lean body mass, or statutorygrowth approximating the normal range, or by other criteria formeasuring growth has defined herein as are deemed appropriate by thepractitioner.

The IGF-I and GH are also suitably administered by sustained releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et. al., Biopolymers, 22, 547-556(1983)), poly(2-hydroxyethyl metharcylate) (R. Langer et. al., J.Biomed. Mater. Res., 15: 167-277 (1981), and R. Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et. al., Id.) orpoly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release IGF-Icompositions also include liposomally entrapped IGF-I. Liposomescontaining IGF-I are prepared by methods known per se: DE 3,218,121;Epstein et. al., Porc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985);Hwang et. al., Proc. Natl. Acad. Sci. U.S. A., 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily, the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. percent cholesterol, the selected proportion beingadjusted for the optimal IGF-I and GH therapy.

For parenteral administration, in one embodiment, the IGF-I and GH areformulated generally by mixing each at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the IGF-I and GHeach uniformly and intimately with liquid carriers or finely dividedsolid carriers or both. Then, if necessary, the product is shaped intothe desired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeprides; proteins, such aseerurn albumin, gelatin, or immunoglobulins; hydrophilic polymers suchas polyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; nonionic surfactantssuch as polysorbates, poloxamers, or PEG; and/or netural salts, e.g.,NaCl, KCl, MgCl₂, CaCl₂, etc.

The IGF-I and GH are each typically formulated individually in suchvehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably1-10 mg/ml, at a pH of about 4.5 to 8. Full-length IGF-I is generallystable at a pH of no more than about 6.5; des(1-3)-IGF-I is stable atabout 3.2 to 5; hGH is stable at a higher pH of about 5.5-9. It will beunderstood that use of certain of the foregoing excipients, carrters,2or stabilizers will result in the formation of IGF-I or GH salts.

In addition, the IGF-I and GH, preferably the full-length IGF-I, aresuitably formulated together in a suitable carrier vehicle to form apharmaceutical composition that does not contain cells. In oneembodiment, the buffer used for formulation will depend on whether thecomposition will be employed immediately upon mixing or stored for lateruse. If employed immediately after mixing, a mixture of full-lengthIGF-I and GH can be formulated in mannitol, glycine, and phosphate, pH4-6. If this mixture is to be stored, it is formulated in a buffer at apH of about 5-6, such as acetate or citrate, with a surfactant thatincreases the solubility of the GH at this pH, such as 0.1-0.2%polysorbate 20 or poloxamer 188. The final preparation may be a stableliquid or lyophilized solid.

In one particularly preferred embodiment, the composition comprisesIGF-I and GH in a weight ratio of IGF-I:GH of between about 2:1 and100:1 (w/w), about 0.05-0.3 mM of an osmolyte, preferably an inorganicsalt and/or sugar alcohol, about 0.1-10 mg/ml of at least onestabilizer, about 1-5 mg/ml of a surfactant, and about 5 to 100 mM of abuffer at about pH 5-6. The more preferred amounts of IGF-I and GR inthis composition are about 2-20 mg/ml IGF-I and about 0.2-10 mg/ml GR.The more preferred weight ratio of IGF-I:GR is about 3:1 to 50:1, morepreferably about 3:1 to 30:1, and still more preferably about 3:1 to25:1, and most preferably about 5:1 to 20:1.

An "osmolyte" refers to an isotonic modifier or osmotic adjuster thatlends osmolality to the buffered solution. Osmolality refers to thetotal osmotic activity contributed by ions and nonionized molecules to asolution. Examples include inorganic salts such as sodium chloride andpotassium chloride, mannitol, polyethylene glycols (PEGs), polypropyleneglycol, glycine, sucrose, glycerol, amino acids, and sugar alcohols suchas mannitol known to the art that are generally regarded as safe (GRAS).The preferred osmolyte herein is sodium chloride or potassium chloride.

The "stabilizer" is any compound that functions to preserve the activeingredients in the formulation, i.e., GH and IGF-I, so that they do notdegrade or otherwise become inactive over a reasonable period of time ordevelop pathogens or toxins that prevent their use. Examples ofstabilizers include preservatives that prevent bacteria, viruses, andfungi from proliferating in the formulation, anti-oxidants, or othercompounds that function in various ways to preserve the stability of theformulation.

For example, quaternary ammonium salts are useful stabilizers in whichthe molecular structure includes a central nitrogen atom joined to fourorganic (usually alkyl or aryl) groups and a negatively charged acidradical. These salts are useful as surface-active germicides for manypathogenic non-sporulating bacteria and fungi and as stabilizers.Examples include octadecyldimethyl benzylammoniumchloride,hexamethoniurnchloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofstabilizers include aromatic alcohols such as phenol and benzy alcohol,alkyl parabens such as methyl or propyl paraben, and m-cresol. The mostpreferred stabilizer herein is phenol or benzyl alcohol.

The stabilizer is included in astable liquid form of the GH and IGF-Iformulation, but not in a lyophilized form of the formulation. In thelatter case, the stabilizer is present in the bacteriostatic water forinjection (BWFI) used for reconstitution. The surfactant is alsooptionally present in the reconstitution diluent.

The "inorganic salt" is a salt that does not have a hydrocarbon-basedcation or anion. Examples include sodium chloride, ammonium chloride,potassium chloride, magnesium chloride, calcium chloride, sodiumphosphate, calcium phosphate, magnesium phosphate, potassium phosphate,ammonium phosphate, sodium sulfate, ammonium sulfate, potassium sulfate,magnesium sulfate, calcium sulfate, etc. Preferably, the cation issodium and the anion is chloride or sulfate, and the most preferredinorganic salt is potassium chloride or sodium chloride.

The "surfactant" acts to increase the solubility of the IGF-I and GH ata pH of about 4-7. It is preferably a nonionic surfactant such as apolysorbate, e.g., polysorbates 20, 60, or 80, a poloxarner, e.g.,poloxamer 184 or 188, or any others known to the art that are GRAS. Morepreferably, the surfactant is a polysorbate or poloxamer, morepreferably a polysorbate, and most preferably polysorbate 20.

The "buffer" may be any suitable buffer that is GRAS and confers a pH of5-6 on the GH+IGF-I formulation and a pH of about 5-5.5 on the IGF-Iformulation. Examples include acetic acid salt buffer, which is any saltof acetic acid, including sodium acetate and potassium acetate,succinate buffer, phosphate buffer, citrate buffer, or any others knownto the art to have the desired effect. The most preferred buffer issodium acetate, optionally in combination with sodium citrate.

The most preferred composition containing both IGF-I and GH is thefollowing: about 7-10 mg/ml of IGF-I, about 0.2-1.5 mg/ml of GH at aweight ratio of IGF-I:GH of about 3:1 to 20:1, about 5-7 mg/ml of sodiumchloride, about 0.1-3 mg/ml of phenol and/or about 6-10 mg/ml of benzylalcohol, about 1.3 mg/ml of polysorbate, about 2.5-4 mg/ml of sodiumacetate, and about 0.1-1 mg/ml of sodium citrate, pH about 5.4.

The final formulation, if a liquid, is preferably stored at atemperature of about 2°-9° C. for up to about four weeks. Alternatively,the formulation can be lyophilized and provided as a powder forreconstitution with water for injection that is stored as described forthe liquid formulation.

IGF-I and GH to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes).

Therapeutic IGF-I and GH compositions generally are placed into acontainer having a Sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

IGF-I and GH ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous GH solution, and the resulting mixtureis lyophilized. The infusion solution is prepared by reconstituting thelyophilized GH using bacteriostatic Water-for-Injection.

It was found that when whole body weight is to be increased withoutconcomitant increases in kidney or thymus weights, the GH IGF-Iformulation is preferably injected. If, however, the object is to affectthe body composition of the patient or to increase not only whole bodyweight but also selected organs such as the thymus and kidney, forexample, in patients that are immunodeficient (such as AIDS patients) orin patients with kidney disorders (such as ischemic or nephrotoxicdysfunction or chronic or acute renal insufficiency), the GH+IGF-Iformulation is preferably infused to the patient.

The formulation containing both the IGF-I and GH can be made by manydifferent methods. One method comprises mixing an IGF-I-containingcomposition (having osmolyte, stabilizer, and buffer as described below)with a buffered solution comprising GH at a pH about 6 in a dose (mg)ratio of from about 2:1 to 100:1 IGF-I:GH up to a dose no greater thanabout 5 mg/ml of GH. Preferably, this buffered solution contains about1-10 mg/ml of GH in about 5-15 mg/ml of an inorganic salt, about 1-5mg/ml of a stabilizer, about 1-5 mg/ml of a surfactant, and sodiumcitrate buffer at pH about 6. More preferably, the liquid GH formulationcontains about 3-5 mg/ml GH, about 8-9 mg/ml sodium chloride, about 1-3mg/ml phenol, about 1-3 mg/ml polysorbate 20, and about 10mM sodiumcitrate, pH about 6.

The IGF-I-containing solution useful for administering IGF-I separatelyfrom GH and for admixing with the GH solution as described above is asfollows: about 2-20 mg/ml of IGF-I, about 2-50 mg/ml of an osmolyte,about 1-15 mg/ml of at least one stabilizer, and a buffer (preferably anacetic acid salt buffer, and most preferably sodium acetate) in anamount such that the composition has a pH of about 5-5.5. The osmolyte,stabilizer, and buffer, and the preferred compounds within thesecategories are defined above. Optionally, the formulation may alsocontain a surfactant selected from the types described above, preferablyin an amount of about 1-5 mg/ml, more preferably about 1-3 mg/ml.

In a preferred embodiment, the osmolyte is an inorganic salt at aconcentration of about 2-10 mg/ml or a sugar alcohol at a concentrationof about 40-50 mg/ml, the stabilizer is benzyl alcohol, phenol, or both,and the buffered solution is an acetic acid salt buffered solution. Morepreferably, the osmolyte is an inorganic salt, most preferably sodiumchloride.

In an even more preferred formulation, the amount of IGF-I is about 8-12mg/ml, the amount of sodium chloride is about 5-6 mg/ml, the stabilizersare benzyl alcohol in an amount of about 8-10 mg/ml and/or phenol in anamount of about 2-3 mg/ml, and the buffer is about 50 mM sodium acetateso that the pH is about 5.4. Optionally, the formulation containspolysorbate as a surfactant in an amount of about 1-3 mg/ml. A 50-mMacetate concentration in the starting IGF-I solution before mixing withGH ensures that the final pH will not vary significantly from 5.4 in thefinal IGF-I/GH mixture to maintain good solubility of both proteins overa wide mixing ratio range. However, a broader pH range in terms ofstability of both proteins is from about 5 to about 6.

The IGF-I formulation of the invention can be used to treat anycondition that would benefit from treatment with IGF-I, including, forexample, diabetes, chronic and acute renal disorders, such as chronicrenal insufficiency, necrosis, etc., obesity, hyperinsulinemia,GH-insufficiency, Turner's syndrome, short stature, undesirable symptomsassociated with aging such as increasing lean mass to fat ratios,immuno-deficiencies including increasing CD4 counts and increasingimmune tolerance, catabolic states associated with wasting, etc., Larondwarfism, insulin resistance, and so forth.

This IGF-I formulation especially was found by itself to have increasedpotency in treating mammals, especially humans, with hyperglycemicdisorders by reducing their glucose levels. It was also found toincrease the mammal's absorbance of the IGF-I if administeredsubcutaneously. For purposes herein, "hyperglycemic disorders" refers toall forms of diabetes, such as type I and type II diabetes, as well ashyperinsulinemia and hyperlipidemia, obese subjects. The preferreddisorder is diabetes, especially type II diabetes.

For treating these various conditions, IGF-I is administered by anysuitable means, including intravenously, intraperitoneally,subcutaneously, or intramuscularly. The effective amount of IGF-I forthis purpose is generally adjusted in accordance with many factors,including the patient's specific disease, the route of administration,the individual weight and general condition of the patient to betreated, and the judgment of the medical practitioner. Caution must betaken to monitor blood glucose periodically to avoid hypoglycemia.

Generally, the dosing will range between about 1 μg/kg/day and up toabout 100 mg/kg/day, preferably 10 μg/kg/day and up to about 10mg/kg/day. If given continuously, the IGF-I is generally administered indoses of about 1 μg/kg/hour up to about 100 μg/kg/hour, either by twodaily injections or by subcutaneous infusions, e.g., via minipump or aportable infusion pump. Preferably, the IGF-I is given subcutaneously orintravenously, and most preferably subcutaneously.

If the IGF-I is administered together with insulin, the latter is usedin lower amounts than if used alone, down to amounts which by themselveshave little effect on blood glucose, i.e., in amounts of between about0.1 IU/kg/24 hour to about 0.5 IU/kg/24 hour.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areexpressly incorporated by reference.

EXAMPLE I I. Protocol

Hypophysectomized adult male rats weighing 85 to 105 grams (Taconic, NY)were received 7 days after surgery and then weighed every 2-3 days forten days meet entry criteria of a weight gain of less than 7 grams andno overall body weight loss. The rats were maintained on Purina rat chowad lithium. Each lot of animals was divided into a control (excipient),a IGF-I-supplemented group, a des(1-3)-IGF-I-supplemented group, aGH-supplemented group, a IGF-I/GH-supplemented group, and ades(1-3)-IGF-I/GH-supplemented group.

Alzet osmotic pumps (Alza, Palo Alto, CA) were implanted to deliverycontinuously either excipient (10 mM citrate buffer and 126 mM NaCl, pH6.0 ) or recombinant human IGF-I (produced in E. coli as a Z--Z fusionpolypeptide by the process generally described in EP 230,869 publishedAug. 5, 1987, or available commercially from KabiGen AB, Stockholm,Sweden (specific activity>14,000 U/mg by radio receptor assay usingplacental membranes), or available for clinical investigations fromGenetech, Inc., South San Francisco). The IGF-I was dissolved at 5 mg/mlin 10 mM citrate buffer and 126 mM NaCl, pH 6.0 and delivered to therats at a rate of 120 μg/rat per day (equivalent to 1.2 mg/kg/dayassuming that the rats weight 100 g each). This rate represents asubmaxial dose that gives a consistent body weight gain in this model.

Alternatively, the pumps were implanted to deliver continuouslyrecombinant human des(1-3)-IGF-I (produced in E. coli as generallydescribed by PCT WO 87/01038 published Feb. 26, 1987 and expected tohave a specific activity of >about 14,000 U/mg by radio receptor assayusing placenta membranes, or available as brain IGF from KabiGen AB,Stockholm, Sweden,×14,000 U/mg by radio receptor assay using placentamembranes). It was then formulated at 2 mg/ml in 20 mM acetic acid, pH3.2, and delivered at a rate of 0.055, 0.166, or 0.5 mg/kg/day.

To the GH-supplemented groups was delivered recombinant methionyl humangrowth hormone (PROTROPIN® brand, Genetech, Inc., South San Francisco,CA) dissolved at 2 mg/ml in 16 mg/ml mannitol and 5 mM phosphate, pH7.8, as excipient. The hGH was injected subcutaneously each day, also atsubmaximal doses (15, 60, and 240 μg/kg per day) for the weight gainresponse. Moore et. al., supra.

Alternatively, recombinant (metless) human growth hormone (Nutropin®brand, Genetech, Inc.) may be employed that is formulated at 2 mg/ml in18 mg/ml mannitol, 0.68 mg/ml glycine, and 5 mM phosphate, pH 7.4.

At pump implant the animals received oxytetracycline in a singleintraperiotoneal injection as an intravital marker to longitudinal bonegrowth.

The growth rates of the hypophysectomized animals were determined byfollowing daily body weights, organ weights at sacrifice, and tibialbone fixed for subsequent assessment of the growth plate. The bone wasdecalcified, bisected longitudinally, and embedded in paraffin forsectioning and staining with toludine blue. The distance between thegerminal cell layer and the transition from active chondocytes to newbone deposits was measured microscopically with the aid of a calibratedocular micrometer. In addition, undecalcified sections were preparedfrom the proximal tibia and the distance between the growth plate andthe tetracycline line, laid down in calcified bone, was determined toassess cumulative longitudinal bone growth.

The remaining solution was removed from all osmotic pumps, and verifiedby immunoassay to contain either excipient, IGF-I, or des(1-3)-IGF-I.Furthermore, the amount of hormones remaining in the pump of each ratwas that expected for continuous delivery over seven days at the rate ofdelivery specified by the manufacturer.

Independent replicate studies are designated as Study 1 and Study 2,performed a month apart. Statistical comparisons were made by analysisof variance with follow-up comparisons made by Duncan's Multiple RangeTest. A p value of less than 0.05 was considered significant. All dataare represented as the mean±SD of 6-8 animals per group. Two otherindependent studies confirmed these data.

II. RESULTS

FIGS. 1A and 1B represent the cumulative daily body weight incrementsfor the hyphohysectomized rats treated with either excipient, 60μg/kg/day hGH, 1.2 mg/kg/day IGF-I, or the hGH/IGF-I combination forseven days for Studies 1 and 2, respectively. The mean±SD of 7-9animals/group is shown in the graphs; statistical significance wasassumed if p>0.05. The excipient control group did not gain or lose asignificant amount of weight during the week, confirming thecompleteness of the hypophysectomy and the health of the animals in bothstudies. The mean body weight was increased by hHG in a dose-dependentmanner such that on days 3-7 the response to all hGH doses weresignificantly different from each other (see FIG. 7). Likewise, IGF-Iproduced a significant body weight gain that was first recognized on day2 of dosing, and by day 7 was highly significantly different fromexcipient (2.9±3.5 g vs. 16.6±2.5 g, t=16.86, p>0.001).

The combination of hGH plus IGF-I yielded a body weight gain that wasgreater than either hormone alone and appeared to be at least additive.By day 7, the body weight increments for the excipient control, IGF-I,hGH, and combination treatments were, respectively: Study 1: 2.91±3.51g, 16.6±2.5 g, 12.9±1.2 g, and 22.2±2.7 g; Study 2: -0.04±2.41 g, 10.8±3g, 9.04≅0.92 g, and 19.3±1.6 g. The weight increment of the combinationgroup was statistically different from the means of the other threegroups. For example, in Study 1 the mean weight gain at day 7 for thecombination (22.2±2.7 g) was greater than that for GH alone (12.9±1.2 g,t=10.80, p<0.001) or for IGF-I alone (16.6±2.5 g, t=6.710, p<0.001). Inthe same experiment (data not shown on this FIG. 1), des-(1-3)-IGF-Ialso increased weight gain (to 19.9±2.6 g), which on the addition of GHwas increased to 24.7±1.3 g (t=5.75, p<0.001).

In contrast, it was reported earlier that when native bovine GH (bGH)was delivered intravenously for four days to hypophysectomized rats, andthen bGH plus methionine-IGF-I for four more days, there was no greaterweight gain than that measured with bGH alone. Skottner, J. End. Crin.,supra. Beyond the different delivery routes and dosing regimens of thesetwo studies, the methionyl-IGF-I itself produced no incremental weightgain in this earlier report. To the contrary, this experiment showsrepeatedly that IGF-I and des(1-3)-IGF-i promote body weight gain inhypophysectomized rats and that there was an additive effect when GH wasco-delivered.

In the hypophysectomized rat weight gain assay, there is an excellentcorrelation between the weight gain and the bone growth responses to GH.Therefore, an enhanced weight gain is likely to be accompanied byenhanced bone growth, as is the case below.

FIG. 2 illustrates a bar=graph of the increase in width of theepiphyseal bone growth plate after seven days of hGH and/or IGF-Itreatment in hypophysectomized rats. The mean±SD for 7-9 rats per groupis illustrated for Study 1. Statistically significant differences wereassumed if p<0.05.

In Study 2, shown in FIG. 3B, the groups treated with 60 μg/kg/day of GH(315±35 μm) or with 120 μg/rat of IGF-I (284±20 μm) were significantlydifferent (t=6.859, p<0.001; t=4.00, p<0.01, respectively) from theexcipient group (235±36 μm); the plate width for GH plus IGF-I group(351±29 μm) differed from both the GH alone (t=3.069, p<0.05) and IGF-Ialone (t=5.535, p<0.001). Thus, in both studies GH and IGF-I aloneinduced a significant widening of the tibial epiphysis as compared tothe control group, whereas co-treatment with both hormones produced agreater width than treatment with either GH or IGF-I by itself, exceptat the high-dose GH level. In addition (FIG. 3B), des(1-3)-IGF-I alsostimulated growth plate width to 300±117 μm compared to excipient(t=5.545, p<0.001), and once again co-administration of GH resulted in afurther increase in plate width to 364±31 μm, which was greater than fordes(1-3)-IGF-I alone (t=5.507, p <0.001) and GH alone (t=4.193, p<0.01).The epiphyseal cartilage widening in response to these hormonetreatments was similar in pattern no the body weight changes (FIG. 1).

As with body weight gain, other investigators have tested the effects ofsuch combination treatments on tibial bone growth. GH and IGF-I,delivered intravenously to rats by Skottner et. al., J. Endocrin.,supra, induced into significantly greater response on tibial bone growthor epiphyseal cartilage width than that resulting from treatment witheither hormone alone. The IGF-I did induce widening of the epiphysealcartilage and lengthening of the bone, while having no effect on bodyweight, as noted above. In another experiment, direct administration ofeither of these hormones to the tibial epiphysis stimulated longitudinalbone growth. Isgaard et. al., supra. However, the combination of IGF-Iand GH yielded no greater growth than that achieved with GH alone.

FIG. 3 illustrates two measures of bone growth, longitudinal bone growth(FIG. 3A) and epiphyseal plate width (FIG. 3B, Study 2 as opposed toStudy 1 shown in FIG. 2, where only epiphyseal plate width is shown),obtained in hypophysectomized rats treated for 7 days with IGF-I ordes(1-3)-IGF-I alone or in combination with hGH. For both full-lengthIGF-I and des(1-3)-IGF-I, the results show that their combination withhGH yielded bone growth or cartilage expansion that was greater than theeffect using either hormone alone and was additive.

The relevant changes in the weights of the five organs measured are asfollows (Table 1). While GH inconsistently increased heart, thygnus, andspleen, IGF-I and the combination of IGF-I and GH clearly increased allorgan weights relative to the excipient group. The preferential effectof IGF-I on kidney, spleen, and thymus has been shown by others. Guleret. al., Proc. Natl. Acad. Sci. USA, 85: 4889-4893 (1988). Asignificantly greater effect of the combination treatment was measuredonly in Study 2, for all organs except the thymus. Correcting for thebody weight increment, the organ-to-body weight ratios were increased byIGF-I for kidneys, spleen, and thymus; the hormone combination did notamplify this effect in these three responsive tissues. In contrast, GHtreatment did not alter the organ-to-body weight ratios.

These data indicate that at least a fraction of the hormone combinationresponse can be attributed to weight increases in

                                      TABLE 1                                     __________________________________________________________________________    GH AND IGF-I ELICIT DIFFERENT ORGAN WEIGHT RESPONSES                                         GH      IGF-I                                                          Excipient                                                                            60 ug/kg                                                                              1.2 mg/kg                                                                             GH + IGF-I                                     __________________________________________________________________________    A. Absolute Wet Weights                                                       Study 1                                                                       Heart (mg)                                                                             291 ± 20                                                                          324 ± 13#                                                                          341 ± 24#                                                                          344 ± 16#                                  Kidneys (mg)                                                                           650 ± 46                                                                          686 ± 60.sup.ab                                                                    849 ± 50#.sup.a                                                                    869 ± 31#.sup.b                            Liver (g)                                                                             3.80 ± 0.17                                                                       4.00 ± 0.23.sup.ab                                                                 4.43 ± 0.27#.sup.a                                                                 4.44 ± 0.39#.sup.b                          Spleen (mg)                                                                            234 ± 56                                                                          244 ± 26.sup.ab                                                                    369 ± 50#.sup.a                                                                    389 ± 54#.sup.b                            Thymus (mg)                                                                            233 ± 24                                                                          317 ± 82#.sup.a                                                                    391 ± 49#                                                                          414 ± 110#.sup.a                           Study 2                                                                       Heart (mg)                                                                             355 ± 22                                                                          374 ± 43.sup.a                                                                     376 ±  24.sup.b                                                                    440 ± 65#.sup.ab                           Kidneys (mg)                                                                           688 ± 37                                                                          736 ± 44.sup.ab                                                                    871 ± 62#.sup.ab                                                                   973 ± 45#.sup.bc                           Liver (g)                                                                             3.77 ± 0.25                                                                       4.04 ± 0.30.sup.a                                                                  4.42 ± 0.41#                                                                       4.58 ± 0.13#.sup.a                          Spleen (mg)                                                                            197 ± 16                                                                          260 ± 24#.sup.a                                                                    297 ± 30#.sup.b                                                                    342 ± 23#.sup.ab                           Thymus (mg)                                                                            257 ± 42                                                                          336 ± 50                                                                           436 ± 154#                                                                         450 ± 113#                                 B. Organ to Body Weight (BW) Ratio (× 10.sup.-3)                        Study 1                                                                       Heart/BW                                                                              3.00 ± 0.19                                                                       3.08 ± 0.17                                                                        3.10 ± 0.21                                                                        2.95 ± 0.15                                 Kidneys/BW                                                                            6.71 ± 0.52                                                                       6.51 ± 0.57.sup.ab                                                                 7.70 ± 0.38#.sup.a                                                                 7.45 ± 0.38#.sup.b                          Liver/BW                                                                              39.2 ± 1.8                                                                        37.9 ± 1.7                                                                         40.2 ± 2.1                                                                         38.0 ± 2.2                                  Spleen/BW                                                                             2.42 ± 0.65                                                                       2.31 ± 0.27.sup. ab                                                                3.35 ± 0.47#.sup.a                                                                 3.33 ± 0.42#.sup.b                          Thymus/BW                                                                             2.41 ± 0.28                                                                       3.00 ± 0.72                                                                        3.55 ± 0.97#                                                                       3.55 ± 0.97#                                Study 2                                                                       Heart/BW                                                                              3.91 ± 0.22                                                                       3.72 ± 0.34                                                                        3.69 ± 0.25                                                                        3.98 ± 0.51                                 Kidneys/BW                                                                            7.57 ± 0.27                                                                       7.33 ± 0.30.sup.ab                                                                 8.56 ± 0.64#.sup.a                                                                 8.80 ± 0.37#.sup.b                          Liver/BW                                                                              41.4 ± 2.2                                                                        40.2 ± 1.9                                                                         43.4 ± 4.2                                                                         41.4 ± 1.0                                  Spleen/BW                                                                             2.16 ± 0.13                                                                       2.69 ± 0.52#.sup.a                                                                 2.92 ± 0.28#                                                                       3.09 ± 0.21#.sup.a                          Thymus/BW                                                                             2.83 ± 0.43                                                                       3.35 ± 0.48                                                                        4.28 ± 1.48#                                                                       4.06 ± 0.99#                                __________________________________________________________________________     Mean ± SD (7-9 rats/group): the # denotes statistically different from     excipient and similar letter superscripts denote group differences by         Duncan's test after analysis of variance (ANOVA) at p < 0.05             

specific organs. In addition, they indicate that the additive effect ofIGF-I and GH was not seen on all tissues, for example, for the absoluteweight of thymus (Table 1), or for all the organ/body weight ratios.This varying sensitivity of different tissues to the combination of GHand IGF-I was unexpected. In some tissues, notably in whole body growthand on bone and cartilage, IGF-I and GH are both effective and additive.In other tissues, i.e., thymus, IGF-I and GH are both effective but notadditive, indicating a selective effect.

EXAMPLE II A. Combination Studies

In the two experiments described below, hypophysectomized rats asdescribed in Example I (Study 3) or female dwarf rats (60-70 days ofage, 100-140 g, Study 4) were anesthetized with ketamine/xylazine. Then2 (for the dwarf rats) or 2 (for the hypophysectomized rats) osmoticminipumps (Alza 2001, delivery rate 1 μl/hour/pump) were placedsubcutaneously. The pumps contained either the excipient (10 mM citratebuffer and 126 mM NaCl, pH 6) or IGF-I (5 mg/ml) so that the approximatedose administered was 240 μg/rat/day (2.4 mg/kg assuming a 100 g rat)for both types of rats. The hGH formulation employed was that describedin Example I. The IGF-I was prepared by direct 8secretion of the IGF-Igene from E. coli as in accordance with EP 128,733 published Dec. 19,1984 or EP 288,451 published Oct. 26, 1988, and expected to have aspecific activity of > about 14,000 U/mg by radio receptor assay usingplacental membranes, or was obtained from KabiGen AB (specificactivity >14,000 U/mg) or from Genentech, Inc. as described in ExampleI. It was formulated as described in Example I. In Study 3 thesolubility of hGH was increased by adding 0.1% Tween 20 to the 5 mMphosphate buffer (pH 7.8). The hGH in both studies was given daily as asingle 0.1-ml subcutaneous injection.

In Study 3 (hypophysectomized rats) the experimental groups were:

1) Excipient pump, excipient injections

2) IGF-I pump (2.4 mg/kg), excipient injections

3) Excipient pump, hGH injections (50.0 mg/kg)

4) Excipient pump, hGH injections (10.0 mg/kg)

5) Excipient pump, hGH injections (2 mg/kg)

6) Excipient pump, hGH injections (0.4 mg/kg)

7) Excipient pump, hGH injections (0.08 mg/kg)

8) IGF-I pump (2.4 mg/kg), hGH injections (50.0 mg/kg)

9) IGF-I pump (2.4 mg/kg), hGK injections (10.0 mg/kg)

10) IGF-I pump (2.4 mg/kg), hGH injections (2.0 mg/kg)

11) IGF-I pump (2.4 mg/kg), hGH injections (0.4 mg/kg)

12) IGF-I pump (2.4 mg/kg), hGH injections (0.08 mg/kg).

In Study 4 (dwarf rate) the experimental groups were:

1) Excipient pump, excipient injections

2) IGF- I pump (2.4 mg/kg) , excipient injections

3) Excipient pump, hGH injections (2.0 mg/kg)

4) Excipient pump, hGH injections (0.5 mg/kg)

6) Excipient pump, hGH injections (0.125 mg/kg)

7) IGF-I pump (2.4 mg/kg), hGH injections (2.0 mg/kg)

8) IGF-I pump (2.4 mg/kg), hGH injections (0.5 mg/kg)

9) IGF-I pump (2.4 mg/kg), hGH injections (0.125 mg/kg).

FIG. 4 shows the results from Study 3 for the 7-day weight gains in thehypophysectomized rat. The excipient gave a weight gain of 4.46+1.66 gand IGF-I at 240 μg/day gave a weight gain of 8.23+1.98 g. Once more,the inclusion of IGF-I in the minipumps greatly enhanced the potency ofdaily injections of hGH in promoting weight gain. The weight gainresponses to hGH or hGH plus IGF-I were analyzed as a parallel linebioassay against log dose of hGH. The two dose response lines fulfilledthe criteria for a bioassay, as they were statistically proved to belinear and parallel. The potency of hGH plus IGF-I was 26.6 times thatof hGH alone (95% confidence, 14.8 to 51.7), with the difference betweenthe two dose-response lines being highly significant (1.49 degrees offreedom (d.f.), F=169.4, p<0.0001).

FIG. 5 shows the weight gains over 7 days from Study 4. The excipientgave a weight gain of 3.95-3.56 g and IGF-I at 240 μg/day gave a weightgain of 12.15±3.76 g. The weight gain responses to hGH or hGH plus IGF-Iwere analyzed as a parallel line bioassay against log dose of hGH. Thetwo dose response lines fulfilled the criteria for a bioassay, as theywere statistically proved to be linear and parallel. Individually, IGF-Iand hGH gave substantial weight gains in the dwarf rat. The relativepotency of the hGH plus IGF-I was 28.9 times that of the hGH alone (95%confidence limits, 7.7 to 514.6), with the difference between the twodose-response lines being highly significant (1,30 d.f., F=45.75,p<0.0001).

B. Dose Response Curve of IGF-I Alone

FIG. 6 illustrates the weight gain of hypophysectomized rats treatedwith excipient (citrate buffer as described above), or the IGF-I ordes(1-3)-IGF-I used in Example I at three different doses subcutaneouslyusing minipumps for seven days, following the general protocol describedin Examples I. This figure illustrates the minimal doses of IGF-I anddes(1-3)-IGF-I for bioactivity in the rat.

C. Dose Response Curve of hGH Alone

FIG. 7 illustrates the weight gains of hypophysectomized rats treatedwith excipient or three different doses of the hGH of Examples I and IIdaily subcutaneously for seven days, following the general protocoldescribed in Example I. This figure illustrates the minimal doses of GHfor bioactivity in the rat. At day 7, low-dose GH showed a greaterweight gain than excipient (2.9 ±3.5 g rs. 8.6±2.3 g, t=7.03, p<0.001),which was in turn less than medium-dose GH (12.9±1.2 g, t=4.91, p<0.01).

In the two animal models of GH deficiency (Studies 3 and 4), the potencyof hGH administered as a daily subcutaneous injection was increased over25 fold by co-treatment with IGF-I. This result in the hypophysectomizedrat might be explained by the relative lack of hormones (thyroid andglucocorticoids) known to be permissive for hGH action leading to a poorIGF-I generation. However, the result in the dwarf rat, where only hGHappears to be lacking, with all the other hormone systems (especiallythe thyroid and adrenal hormones) being normal, indicates that theadditive effect of hGH and IGF-I occurs independent of the status ofthyroid or adrenal hormones. However, the close agreement in the twomodels of the enhanced potency of hGH due to IGF-I and the magnitude ofthe effect (about 25X) is surprising.

The doses of hGH that were used in Study 3 have rarely been used in thehypophysectomized rat, and the literature is unclear as to the dose ofhGH that gives a maximal growth response. Doses of 10 and 50 mg/kg/daygiven as single daily subcutaneous injections for one week produce amaximal growth response. But the dose responses for the two regimes (hGHand hGH plus IGF-I) were parallel, even over this 625-fold dose range offive doses of hGH, including the two maximal doses of hGH. Therefore,the maximal growth response to hGH can clearly be increased if IGF-I isco-administered. This is surprising, as the maximal weight gain responseto IGF-I in the hypophysectomized rat appears to be less than the weightgain in response to hGH.

The range of doses of hGH over which IGF-I would be predicted to have anadditive effect on weight gain is clearly the full range of effective GHdose, in the hypophysectomized rat from 0.01 to 50 mg/kg. In the dwarfrat the maximal effective doses of hGH are not known, but 50 mg/kg wouldalso be assumed to be an effective maximal dose of hGH. The previouswork in the hypophysectomized rat has shown 2.4 mg/kg of IGF-I deliveredas a subcutaneous infusion for one week to be near to maximal, as higherdoses of IGF-I cause fatal hypoglycemia. The minimal effective dose ofIGF-I in the hypophysectomized rat is around 0.1 mg/kg per day.

In the dwarf rat, 2.4 mg/kg of IGF-I was used, while in thehypophysectomized rat both 1.2 mg/kg and 2.4 mg/kg doses of IGF-I wereused (Examples I and II), yet an additive effect of IGF-I and GH wasobserved despite different doses of IGF-I being used. The full doseresponse curves for GH alone and GH plus IGF-I were parallel, whichimplies that at any dose of hGH, even at a very small dose of hGH thatby itself might not give a measurable response, the effects of IGF-I andGH would be additive. It would therefore be expected that at any dailydose of GH (from 0.01 to 50 mg/kg) or IGF-I (from 0.1 to 2.4 mg/kg) thetwo molecules would have additive effects on body growth.

EXAMPLE III Two Clinical Scenarios for the Combination Treatment

Two examples of pertinent clinical scenarios are described below thatwill undoubtedly benefit from concomitant administration of GH andIGF-I.

1. Patients who exhibit a slowing in growth rate after at least twelvemonths of GH administration.

It is well recognized by pediatric endocrinologists that either naive(no previous treatment) or previously treated patients (following abreak in GH administration) exhibit a second-year fall in growth rate.This phenomenon is independent of the etiology of the type of shortstature or GH deficiency (e. g. , whether idiopathic, organic,septo-optic dysplasia (S-O D) , Turner, or other). See FIG. 8.

Thus, during the period where the growth rate is slowing, IGF-Itreatment together with GH treatment would increase the annualized rateto compensate for this second-year loss in response.

2. Patients who have little time for GH administration to be maximallyeffective.

If patients are older when they are diagnosed with GH deficiency, lesstime is available to correct their resultant short stature. This isillustrated in FIG. 9, where the annualized growth rate is reported forpatients in seven age groups. Older patients have only, for example, 2-3years left before their growth plates close, making further lineargrowth unlikely. These patients could be treated with the combination ofIGF-I and hGH to allow optimization of their growth rates.

DISCUSSION AND SUMMARY

The results shown herein have significance in medicine and agriculturein any situation where GH or IGF-I treatment is used. This regime ofcombined IGF-I and GH treatment would allow smaller doses of GH(approximately 25-fold less) to be given to produce equivalent responsesto treatment with GH alone. This would be of particular importance insituations where the side effects of GH treatment (i.e.,hyperinsulinemia, hyperglycemia) should be minimized. In diabetes,combined GH and IGF-I treatment, with smaller GH doses being possible,would minimize the insulin-resistant effect of the administered GH. Inpatients where the anabolic effect of GH is reduced, possibly by areduced ability to produce an IGF-I response to the administered GH,co-treatment with GH and IGF-I would also be expected to give a largeranabolic response.

A broad class of patients where the regime of combined GH and IGF-Itreatment would be beneficial is in adult patients where the IGF-Iresponse to GH is naturally reduced. In adults, the unwanted effects ofGH (insulin resistance) may be a direct consequence of a reduced IGF-Iresponse to administered GH. In adults, the co-administration of GH andIGF-I might be viewed as restoring the situation in a younger animalwhere there is a more vigorous IGF-I response to GH treatment.

The mode of administration of the GH in the present studies wasintermittent, by daily subcutaneous injection. However, at the largestdoses used (50 mg/kg), considerable concentrations of hGH would havepersisted in the blood at physiologically effective concentrations,making the blood concentrations of hGH always at a level that wouldprovide a stimulus to GH receptors. Therefore, at the highest dose thetissue exposure to hGH was in essence one of continuous exposure, sothat the growth response to administering hGH as a continuous infusionwould likely be enhanced by the co-adiministration of IGF-I. The potencyof hGH delivered in any manner that would stimulate body growth or beanabolic would be expected to increase if IGF-I were co-administered.Also, it is likely that the improved potency of co-administered hGH andIGF-I would allow less frequent injections of hGH or IGF-I than for hGHalone.

IGF-I was delivered as a continuous infusion, because previous studiesshowed that IGF-I given alone as injections is less effective atenhancing body growth. However, the combination of GH plus IGF-I wouldallow the use of sub-optimal regimes of IGF-I administration, such asinjections, when combined with GH treatment.

In conclusion, cotreatment of hypophysectomized or dwarf rats with GHand IGF-I or des(1-3)-IGF-I amplifies the body weight gain, longitudinalbone growth, and tibial epiphyseal widening relative to the response toeither hormone alone. This finding indicates for the first time thatexogenous IGF-I can increase some growth responses initiated by GH in amanner that is at least additive. Thus, the IGF-I is effective atincreasing the responses to GH treatment or at decreasing the amount ofGH needed to produce a significant response.

EXAMPLE IV Preparation of IGF-I Formulation and Combination of IGF-I andGH

It was desired to produce a formulation of IGF-I that could be mixedwith hGH in dose ratios of IGF-I:hGH of greater than about 2:1 toprovide a stable co-mix of both proteins. In this example, the IGF-Iformulation used to achieve this was:

10 mg/ml IGF-I

5.84 mg/ml NaCl

9.0 mg/ml benzyl alcohol

2.0 mg/ml polysorbate 20

50 mM sodium acetate pH 5.4.

The intended final product configuration contained 7 ml (70 mg) of theabove solution in a 10-ml glass vial, which is generally storedrefrigerated (2°-8° C.) to maximize its lifetime. This product isdesigned to be a ready-to-use liquid for subcutaneous or intravenousadministration using a conventional needle and syringe.

For administration of GH and IGF-I together rather than separately, theabove formulation (70 mg IGF-I vial) was mixed with a liquid formulationof hGH (5 mg/ml hGH, 8.77 mg/ml NaCL, 2.5 mg/ml phenol, 2.0 mg/mlpolysorbate 20, and 10 mM sodium citrate, pH 6.0), available fromGenentech, Inc. The hGH was added up to about 10 mg (2 ml) hGH. Theformulations were mixed in dose ratios of 7:1, 14:1, and 28:1 IGF-I:hGH.The resulting formulations were generally stored at 2°-8° C. and usedwithin a two-week period. These final formulations had concentrationranges as follows:

IGF-I 7.1-9.6 mg/ml

hGH 0.2-1.4 mg/ml

NaCl1 6.0-6.7 mg/ml

Phenol 0.1-0.7 mg/ml

Benzyl alcohol 6.4-8.7 mg/ml

Polysorbate 20 2.0 mg/ml

Sodium citrate 0.1-0.7 mg/ml

Sodium acetate 2.8-3.8 mg/ml

The aim of the metabolic studies reported in Examples V-XIII below wasto investigate the hypoglycemic effects of different formulations ofrecombinant had IGF-I and their pharmacokinetics, alone or incombination with GH, on plasma glucose levels in the anesthetized dwarf(dw/dw) rat or the normal rat. The aim of the growth study reported inExample XIV below was to investigate the anabolic effect of aformulation containing the combination of GH and IGF-I in the dw/dw ratand to compare this effect with that of formulations with either agentalone.

EXAMPLE V

The fall in blood glucose caused by an injection of IGF-I is a rapidresponse that can be easily measured and can serve as a reasonablebioassay for the "insulin-like" activity, or bioactivity in vivo, ofIGF-I.

It had been found in the dw/dw rat that doses of 750 and 250 μg of IGF-Iformulated in a citrate buffer at pH 6.0 and given subcutaneouslyreduced blood glucose by a moderate and maximal amount, respectively.Therefore, in this Example these doses of IGF-I were given.

EXPERIMENTAL DESIGN

Sixteen 11-week-old female dw/dw rats (138-162 g; Simonsen Labs. GilroyCA) were anesthetized using Ketamine (62.5 mg/kg)/Rompun® Xylazine (12.5mg/kg) anesthesia, intraperitoneally. An additional dose was given asneeded to maintain anesthesia throughout the study. The right jugularvein was cannulated using Microrenathane® 0.033 OD×0.014 ID (BraintreeScientific, Braintree, MA) inserted 23 mm into the jugular. The free endof the cannula was attached to an automated blood sampling machine tocollect blood samples at various timepoints.

Four treatment groups, four rats per group were dosed subcutaneouslywith one of the four IGF-I formulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl, pH 6.0):

750 μg in 150 μl of solution

250 μg in 150 μl of solution

B. pH 5.0 formulation of IGF-I (10 mg/ml in 20 mM sodium acetate buffer,2.5 mg/ml (0.25%) phenol, 45 mg/ml mannitol, pH 5.0):

3) 750 μg in 150 μl of solution

4) 250 μg in 150 μl of solution

Three blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 20, 40, 60, 80, 100, 120, 150, 180,and 210 minutes post-injection. The plasma was immediately separated bycentrifugation. The glucose concentration in the plasma was subsequentlydetermined by a coupled hexokinase procedure using a Monarch 2000chemical systems instrument. Statistical comparisons were made by ananalysis of variance with a Duncan's Multiple Range test. A p value ofless than 0.05 was considered as being statistically significant. Alldata are represented as the mean±Standard deviation with four animalsper treatment group.

There was considerable variation between animals in their basal bloodglucose, but much less variation within an animal for the three initialblood glucose measurements. Therefore, it was decided to express theblood glucose measurements for each individual animal as a percentage ofthe mean of the three initial pre-injection basal blood glucose valuesof that animal.

FIG. 10 shows the mean percentage changes in blood glucose with timeafter a subcutaneous injection of IGF-I given at time zero. There was aclear dose-related reduction in blood glucose. The 750-μg dose of IGF-Igave equal decreases in blood glucose for both formulations of IGF-I.However, at the lower dose of IGF-I (250 μg) there was a cleardifference between the two preparations of IGF-I that remainedstatistically significant at each time point from 60 to 210 minutesPost-injection. The formulation in sodium acetate at pH 5.0 thereforewas more potent.

EXAMPLE VI

In Example V it was found that the re-formulated IGF-I at pH 5.0appeared to be more potent, as it had a greater effect on blood glucosethan the citrate-buffered formulation of pH 6.0. As the response to the750-μg dose appeared to be maximal, and because the re-formulated IGF-Iwas more potent, the doses of IGF-I were reduced in this example to 450and 150 μg/rat.

Experimental Design

Female dw/dw rats (122-138 g) were anesthetized with ketarnine/Rompun®anesthesia, a jugular catheter was inserted, and blood samples weretaken as described for Example V.

Four treatment groups, four rats per group, were dosed subcutaneouslywith one of the four solutions of two IGF-I formulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl, pH 6.0);

1) 450 μg in 150 μl of solution

2) 150 μg in 150 μl of solution

B. pH 5.0 formulation of IGF-I (10 mg/ml in 20 mM sodium acetate buffer,2.5 mg/ml phenol, 45 mg/ml mannitol, pH 5.0):

3) 450 μg in 150 μl of solution

4) 150 μg in 150 μl of solution

Three blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 20, 40, 60, 80, 100, 120, 150, 180,and 210minutes post-injection. The experiment was otherwise conducted inan identical manner to Example V.

Results

The blood glucose measurements for each individual animal were expressedas a percentage of the mean of the three initial preinjection basalblood glucose values of that animal.

FIG. 11 shows the mean percentage changes in blood glucose with timeafter a subcutaneous injection of IGF-I given at time zero. As inExample V, there was a clear dose-related reduction in blood glucose.The 450-μg dose of IGF-I gave similar initial reductions in bloodglucose for both formulations of IGF-I. However, at later time pointsthe blood glucose values for the pH 6.0 formulation at 450 μg rose abovethose for the pH 5.0 re-formulated IGF-I, although this differenceapproached but did not reach statistical significance (p<0.1). However,at the lower dose of IGF-I (150 μg) there was once more a cleardifference between the two preparations of IGF-I that remainedstatistically significant at each time point from 60 to 210 minutespost-injection.

EXAMPLE VII

In Examples V and VI it was established that the re-formulated pH 5.0IGF-I had an increased potency over the pH 6.0 formulation. The presentexample was performed to determine if this increase in potency was dueto the IGF-I being better absorbed from the subcutaneous injection siteor whether the IGF-I was in some way inherently more bioactive. Bothformulations of IGF-I were given as a 150-μl bolus at 150 μg per doseeither subcutaneously or intravenously. The intravenous injection wasgiven via the jugular catheter and blood samples were taken as describedin Example V.

Experimental Design

Four treatment groups of female dw/dw rats, four rats per group, weredosed as indicated with one of the four solutions of two IGF-Iformulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM Nacl, pH 6.0):

1) 150 μg in 150 μl of solution subcutaneously

2) 150 μg in 150 μl of solution intravenously

B. pH 5.0 formulation of IGF-I (10 mg/ml in 20 mM sodium acetate buffer,2.5 mg/ml phenol, 45 mg/ml mannitol, pH 5.0):

3) 150 μg in 150 μl of solution subcutaneously

4) 150 μg in 150 μl of solution intravenously

Three blood samples were taken before the injections at -15, -10, and -5minutes. Then samples were taken at 5, 10, 15, 20, 30, 60, 90, 120, and150 minutes post-injection, as the instant exposure to an intravenousinjection gives more rapid responses than to a subcutaneous injection.The experiment was otherwise conducted in an identical manner to ExampleV.

Results

The blood glucose measurements for each individual animal were againexpressed as a percentage of the mean of the three initial pre-injectionbasal blood glucose values of that animal.

FIG. 12 shows the mean percentage changes in blood glucose with timeafter a subcutaneous or an intravenous injection of IGF-I given at timezero. In this experiment the results in Example VI were confirmed, asthere was a clear difference between the blood glucose responses to thetwo formulations given subcutaneously at the 150-μg dose of IGF-I.However, the blood glucose values for the two formulations of IGF-I werenearly identical if they were delivered intravenously.

Therefore, a clear difference was seen in the bioactivity of the IGF-Iwhen given subcutaneously, but little or no difference when it was givenintravenously. These data suggest that the difference between the twoformulations was primarily an effect on the amount, or nature, of theIGF-I absorbed into the blood from the site of subcutaneous injection.

EXAMPLE VIII

In Examples V-VII it was established that the pH 5.0 reformulated IGF-Ihad increased potency when given by subcutaneous injection, and thatthis was probably related to an increased absorption of the IGF-I in thepH 5.0 formulation. The present example questions whether this increasein potency was due to the IGF-I being better absorbed from thesubcutaneous injection site at the lower pH (6 versus 5). Therefore, thepH 6.0 formulation of IGF-I in citrate buffer, but at low pHs (dose 150μg), was given subcutaneously. Blood samples were taken as described inExample V.

Experimental Design

Four treatment groups of female dw/dw rats, four rats per group, weredosed subcutaneously with one of the four solutions of two IGF-Iformulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl pH 6.0)

1) 450 μg in 150 μl of solution

2) 150 μg in 150 μl of solution

B. pH 5.0 formulation of IGF-I(5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl pH 5.0):

3) 450 μg in 150 μl of solution

4) 150 μg in 150 μl of solution

Three blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 20, 40, 60, 80, 100, 120, 150, 180,and 210 minutes. The experiment was otherwise conducted in an identicalmanner to Example V.

Results

The blood glucose measurements for each individual animal were againexpressed as a percentage of the mean of the three initial pre-injectionbasal blood glucose values of that animal.

FIG. 13 shows the mean percentage changes in blood glucose with timeafter a subcutaneous injection of IGF-I given at time zero. In thisexperiment there was no clear difference between the blood glucoseresponses to the two formulations given at the dose of IGF-I. However,the blood glucose values for the two formulations of IGF-I given at the450-μg dose appeared to be different at later time points: thisdifference approached statistical significance (p<0.1).

Thus, some evidence was obtained showing that a difference in pH betweenIGF-I formulations could affect the absorption of IGF-I. However,changing the pH of the citrate-buffered formulation from pH 6 to pH 5did not produce the large difference in potency that was seen betweenthe two formulations of IGF-I in Examples V-VII. Therefore, theabsorption of IGF-I from the pH 6.0 formulation can unexpectedly beimproved by a combination of pH and formulation changes.

EXAMPLE IX

In Examples V-VIII it was established that a re-formulation of IGF-Icould change its bioactivity. In this example, a new formulation ofIGF-I is devised that can be co-mixed with hGH. The bioactivity of thisnew IGF-I formulation is tested to assess the effects of a different pH(5.4) and different additives and ions.

The design of Example VI was repeated to discover if there was anincreased potency of the pH 5.4 formulation given subcutaneously and ifthis was due to the IGF-I being better absorbed from the subcutaneousinjection site or whether the IGF-I was in some way inherently morebioactive (tested by intravenous injection). Both formulations of IGF-Iwere therefore given at one dose (150 μg) either subcutaneously orintravenously. The intravenous injection was given via the jugularcatheter and blood samples were taken as described in Example V.

Experimental Design

Four treatment groups of female dw/dw rats, four rats per group, weredosed as indicated with one of the two IGF-I formulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl, pH6.0):

1) 150 μg in 150 μl of solution subcutaneously

2) 150 μg in 150 μl of solution intravenously

B. pH 5.4 formulation of IGF-I (10 mg/ml in 50 mM sodium acetate buffer,2.5 mg/ml phenol, 5.84 mg/ml NaCl and 9 mg/ml benzyl alcohol, pH 5.4):

3) 150 μg in 150 μl of solution subcutaneously

4) 150 μg in 150 μl of solution intravenously

Blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 10, 20, 30, 45, 60, 90, and 120minutes. The experiment was otherwise conducted in an identical mannerto Example V.

Results

The blood glucose levels for each individual animal were expressed as apercentage of the mean of the three initial pre-injection basal bloodglucose values of that animal.

FIG. 14 shows the mean percentage changes in blood glucose with timeafter a subcutaneous injection of IGF-I given at time zero. In thisexperiment samples at 10, 20, and 30 minutes were lost, due tooverdilution, so only the data from 45 minutes onward is shown. In thisexample, there was a clear difference between the blood glucoseresponses to the two formulations given subcutaneously at the 150-μgdose of IGF-I. However, the blood glucose values for the twoformulations of IGF-I given intravenously at the 150-μg dose were notsignificantly different. It can also be seen that the response at 120.minutes for the subcutaneously delivered pH 5.4 formulation IGF-Iapproached that of the intravenously delivered dose.

Therefore, the pH 5.4 IGF-I formulation also was very well absorbedcompared to the pH 6.0 formulation of IGF-I. A comparison of FIGS. 12and 14 indicates that the pH 5.4 formulation used in the present studywas probably superior to the pH 5.0 formulation used in Example VII.Changing the pH Of the formulation from pH 6 to pH 5.4 and changing thecomponents of the formulation unexpectedly led to marked increases inbiopotency.

EXAMPLE X

This example repeats the design of Example IX to attempt to duplicatethe results showing an increased potency of the pH 5.4 formulation givensubcutaneously but a similar effectiveness when given intravenously.Therefore, both formulations of IGF-I were given at one dose (150 μg)either subcutaneously or intravenously. The intravenous injection wasgiven via the jugular catheter and blood samples were taken as describedin Example V.

Experimental Design

Four treatment groups of female dw/dw rats, four rats per group, weredosed with one of the two IGF-I formulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl pH 6.0):

1) 150 μg in 150 μl of solution subcutaneously

2) 150 μg in 150 μl of solution intravenously

B. pH 5.4 formulation of IGF-I (10 mg/ml in 50 mM sodium acetate buffer,2.5 mg/ml phenol, 5.84 mg/ml NaCl and 9 mg/ml benzyl alcohol, pH 5.4):

3) 150 μg in 150 μl of solution subcutaneously

4) 150 μg in 150 μl of solution intravenously

Blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 10, 20, 30, 60, 90, and 120 minutes.The experiment was otherwise conducted in an identical manner to ExampleV. However, in addition to blood glucose levels, the plasma IGF-Iconcentration was measured to determine directly its absorption andclearance from the blood. The IGF-I concentration in the plasma sampleswas measured (after acid-ethanol extraction to remove the IGF bindingproteins) by radioimmunoassay.

Results

This example shows the absolute (FIG. 15) and the mean percentage (FIG.16) changes in blood glucose with time after subcutaneous or intravenousinjections of IGF-I given at time zero. A clear difference existedbetween the blood glucose responses to the two formulation givensubcutaneously at the 150-μg dose of IGF-I. However, the blood glucosevalues for the two formulations of IGF-I given intravenously at the150-μg dose were not significantly different. It can also be seen thatthe response at 60 minutes for the subcutaneously delivered pH 5.4 IGF-Iformulation approached that of the intravenous dosing.

Also shown are the plasma IGF-I concentrations after intravenous (FIG.17) or subcutaneous (FIG. 18) injections of IGF-I given at time zero.There was a clear difference between the plasma IGF-I concentrations forthe two formulations given subcutaneously. The concentration of IGF-Iwas increased by about 40 ng/ml (from about 30 to about 70 ng/ml) by thepH 6.0 formulation, but was increased by about 80 ng/ml (from about 30to about 110 ng/ml) by the pH 5.4 formulation. However, the plasma IGF-Iconcentrations for the two formulations of IGF-I (FIG. 17) givenintravenously were not significantly different.

This experiment confirmed and extended the results of Example IX. Onceagain, the pH 5.4 IGF-I formulation was very well absorbed compared tothe pH 6.0 formulation of IGF-I. The improved absorption is now directlyshown, as the plasma IGF-I concentrations were doubled when the samedose of IGF-I was given subcutaneously in the pH 5.4 formulation. The pH5.4 formulation of IGF-I gave a hypoglycemic response that was nearlyidentical to that of the same dose of IGF,I given intravenously. Thesedata suggest that the IGF-I delivered in the pH 5.4 formulation isnearly 100% bioavailable to the rat. These data confirm that theabsorption of IGF-I from the pH 5.4 formulation is unexpectedly improvedover that from the pH 6.0 formulation by a combination of pH andformulation changes.

Summary

By measuring the hypoglycemic response to intravenous and subcutaneousdosing of the two formulations of the IGF-I, it may be concluded that:

1. The pH 5.4 formulation is more potent and better absorbed than the pH6.0 formulation when given subcutaneously.

2. The two formulations give statistically equivalent hypoglycemicresponses when dosing is intravenous.

3. There is a suggestion that the mean fall in blood glucose is greaterwith the pH 5.4 formulation even with intravenous dosing, although thisdoes not reach statistical significance.

EXAMPLE XI

In Example X it was established that the re-formulated pH 5.4 IGF-I hadan increased potency when given by subcutaneous injection. This examplewas designed to determine the relative potency of the two preparationsof IGF-I by giving two doses of IGF-I by subcutaneous injection andmeasuring hypoglycemia and the serum IGF-I concentrations. It appearedthat the pH 5.4 formulation was about 3-fold more effective as ahypoglycemic agent than the pH 6.0 formulation. Therefore, the pH 6.0formulation of IGF-I was given by subcutaneous injection at two doses(150 μg and 450 μg) and the pH 5.4 formulation of IGF-I at two doses (50μg and 150 μg), such that matching effects on blood glucose and serumIGF-I might be expected. In addition, the rapid absorption of IGF-I wasmeasured by taking samples at very frequent intervals immediatelyfollowing the injection of the respective IGF-I formulations.

Experimental Design

Four treatment groups of female dw/dw rats, four rats per group, wereinjected subcutaneously with one of the four solutions of two IGF-Iformulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl pH 6.0):

1) 150 μg in 100 μl of solution

2) 450 μg in 100 μl of solution

B. pH 5.4 formulation of IGF-I (10 mg/ml in 50 mM sodium acetate buffer,2.5 mg/ml phenol, 5.84 mg/ml NaCl and 9 mg/ml benzyl alcohol, pH 5.4):

3) 50 μg in 100 μl of solution

4) 150 μg in 100 μl of solution

Three blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 3, 6, 9, 20, 30, 45, 60, 90, and 120minutes. The experiment was otherwise conducted in an identical mannerto Example V.

Results

In this example the absolute (FIG. 19) and the mean percentage (FIG. 20)changes in blood glucose with time after subcutaneous injections ofIGF-I given at time zero are shown. There was a clear difference betweenthe blood glucose responses to the two formulations givensubcutaneously. At three-fold different doses of IGF-I, equivalenthypoglycemic responses were obtained, as the 150-μg dose of the pH 6.0formulation and the 50-μg dose of the pH 5.4 formulation wereequivalent, as were the 450-μg dose of the pH 6.0 formulation and the150-μg dose of the pH 5.4 formulation.

FIG. 21 shows the plasma IGF-I concentrations after the subcutaneousinjections of IGF-I given at time zero. The pre-injection IGF-Iconcentrations were not different for the four groups. The IGF-Iconcentrations achieved were dose related and directly mirrored theblood glucose concentrations. At three-fold different doses of IGF-I,equivalent plasma IGF-I concentrations were obtained for the 150-μg doseof the pH 6.0 formulation and the 50-μg dose of the pH 5.4 formulationand for the 450-μg dose of the pH 6.0 formulation and the 150-μg dose ofthe pH 5.4 formulation.

In conclusion, following a subcutaneous injection, the absorption andtherefore the efficacy of the IGF-I was improved about 3-fold using thepH 5.4 formulation of IGF-I.

EXAMPLE XII

The previous examples established that the re-formulated pH 5.4 IGF-Ihad an increased potency in terms of a hypoglycemic response when givenby subcutaneous injection. In addition, the examples showed that after asubcutaneous injection a three-fold increase in the absorption andefficacy of the IGF-I was measured using the re-formulated pH 5.4 IGF-I.

All these experiments were conducted in the dw/dw rat. It is possiblethat such rats, which are GH-deficient and IGF-I-deficient compared to anormal GH-sufficient and IGF-I-sufficient rat, might in some way allowIGF-I to be absorbed much better in the pH 5.4 formulation. In thepresent example, IGF-I was given by subcutaneous injection in a normalrat, and blood glucose and serum IGF-I concentrations were measured. Asthese normal rats weighed twice as much (230-250 grams) as the dw/dwrats, and might be expected to have higher concentrations of plasma IGFbinding proteins, the doses of IGF-I were doubled, compared to thoseused in the earlier examples in the dw/dw rat.

Experimental Design

Four groups of normal male Sprague-Dawley rats (230-250 four rats pergroup, were dosed subcutaneously with one of the four solutions of thetwo IGF-I formulations below:

A. pH 6.0 formulation of IGF-,I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl pH 6.0):

1) 300 μg in 200 μl of solution

2) 900 μg in 200 μl of solution

B. pH 5.4 formulation of IGF-I (10 mg/ml in 50 mM sodium acetate buffer,2.5 mg/ml phenol, 5.84 mg/ml NaCl and 9 mg/ml benzyl alcohol, pH 5.4):

3) 100 μg in 200 μl of solution

4) 300 μg in 200 μl of solution

Three blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 3, 6, 9, 20, 30, 45, 60, and 90minutes. The experiment was otherwise conducted in an identical mannerto Example V.

Results

FIG. 22 shows the mean percentage changes in blood glucose with timeafter a subcutaneous injection of IGF-I given at time zero. There was aclear difference between the blood glucose responses to the twoformulations given subcutaneously at the 300μg dose of IGF-I. Thehypoglycemic response induced by 300 μg of the pH 6.0 formulation ofIGF-I was nearly identical to that induced by 100 μg of the pH 5,4formulation of IGF-I. The response to 900 μg of the pH 6.0 formulationwas also similar to the response to 300 μg of the pH 5.4 formulation ofIGF-I.

Therefore, the pH 5.4 formulation of IGF-I showed improved efficacycompared to the pH 6.0 formulation when injected subcutaneously in anormal rat. Data obtained in GH- and IGF-I-deficient dwarf animals couldthus be extrapolated to a GH- and IGF-I-sufficient normal animal.

EXAMPLE XIII

The previous examples established that the re-formulated pH 5.4 IGF-Ihad an increased potency in terms of a hypoglycemic response when givenby subcutaneous injection in normal rats. After a subcutaneous injectiona three-fold increase in the absorption and efficacy of the IGF-I wasmeasured using the reformulated pH 5.4 IGF-I.

In these normal male rats weighing 230-250 grams it was established thata dose of 300 μg of IGF-I caused a small reduction in blood glucose whengiven in the pH 6.0 formulation or a large reduction when given in thepH 5.4 formulation. An object of this example was to develop aformulation in which IGF-I was stable, and which also could be co-mixedwith hGH.

In this example, therefore,.IGF-I was co-delivered with hGH, and theeffect of the hGH and its formulation on the absorption of the IGF-I wasstudied. The efficacy of the co-mix was tested when given bysubcutaneous injection, the usual route of injection for thesetherapeutic drugs. This example tested the acute hypoglycemic efficacyof the co-mix. In the next example, the long-term anabolic efficacy ofthe co-mix on body growth was tested.

Experimental Design

Four groups of normal mate Sprague-Dawley rats (230-250 g), four ratsper group, were dosed subcutaneously with one of the four IGF-I orIGF-I/GH formulations below:

A. pH 6.0 formulation of IGF-I (5 mg/ml in 10 mM sodium citrate bufferand 126 mM NaCl pHE6.0):

1) 300 μg IGF-I in 200 μl of solution

B. pH 5.4 formulation of IGF-I (10 mg/ml in 50 mM sodium acetate buffer,2.5 mg/ml phenol, 5.84 mg/ml NaCl and 9 mg/ml benzyl alcohol, pH 5.4)and/or pH 6.0 formulation of hGH (5 mg/ml in 10 mM sodium citratebuffer, 2.5 mg/ml phenol, 8.77 mg/ml NaCl and 2.0 mg/ml polysorbate 20,pH 6.0):

2) 300 μg IGF-I in 200 μl of solution

3) 300 μg IGF-I+100 μg hGH in 200 μl of solution

4) 300 μg IGF-I+10 μg hGH in 200 μl of solution

Three blood samples were taken before the injections at -15, -10, and -5minutes; then samples were taken at 3, 6, 9, 20, 30, 45, 60, 90, and 120minutes. The experiment was otherwise conducted in an identical mannerto Example V.

Results

FIG. 23 shows the mean percentage changes in blood glucose with timeafter a subcutaneous injection of IGF-I given at time zero. There was asmall reduction in blood glucose when the pH 6.0 formulation was givenbut a much larger fall when the pH 5.4 formulation-was given. Thehypoglycemia induced by the co-mixes of hGH and IGF-I formulation wassimilar to that induced by the pH 5.4 IGF-I formulation alone. At the60-minute timepoint all three groups receiving the pH 5.4 formulationhad significantly lower blood glucose levels than the group thatreceived the pH 6.0 formulation of IGF-I.

FIG. 24 shows the plasma IGF-I concentrations after the subcutaneousinjections of IGF-I given at time zero. The pre-injection IGF-Iconcentrations were not different for the four groups. The IGF-Iconcentrations tended to mirror the blood glucose concentrations, withthe pH 6.0 formulation of IGF-I tending to be more poorly absorbedcompared to the pH 5.4 IGF-I formulation. The co-mix of hGH and IGF-Iled to similar blood IGF-I concentrations to that of the pH 5.4 IGF-Iformulation alone.

Changing the formulation to one that was mixable with hGH led to thebest IGF-I formulation in terms of hypoglycemic activity and IGF-Iabsorption, even when co-mixed with hGH.

EXAMPLE XIV

The previous examples have shown that the re-formulated pH 5-5.5 IGF-Iformulations enhance the absorption and bioactivity of IGF-I. Inaddition, these re-formulations of IGF-I can be co-mixed with hGH whilemaintaining IGF-I absorption. In this example, the anabolic activity ofthe co-mixed Combination is studied using two doses of hGH and a fixeddose of IGF-I.

In Examples I and II it was established that subcutaneous injections ofhGH and infusions of iGF-I gave additive growth responses in the rat.Now it was desired to known if the co-formulation of IGF-I and hGH couldbe delivered to an animal with the additive anabolic effects of IGF-Iand hGH being retained. The options were to deliver the mixture eitheras an injection (as with hGH) or as an infusion (as with IGF-I).

First, the co-formulation could be delivered as a co-mix by injection.It was shown in Examples I and II that infused IGF-I and injected GH hadan additive effect. Since it was found that infused GH did not have suchan effect with IGF-I, injections were the obvious method of deliveringthe hGH. However, IGF-I had been given by infusion in these examples toshow additive effects. It had not been shown that the pH 6.0 IGF-Iformulation given by injection was efficacious in terms of inducing anadditive anabolic response with co-injected hGH.

Nevertheless, when it was attempted to induce growth responses inGH-deficient rats by delivering the pH 6.0 formulation alone byinjection, it was found that IGF-I injections were very poor at inducingan anabolic effect, but very effective at inducing a hypoglycemicresponse. U.S. Pat. No. 5,187,151. The poor anabolic effect of IGF-Iinjections had also been shown in the mouse. Woodall et. al., Horm.Metab. res., 23: 581-584 (1991). Four daily injections of IGF-I (twodaily injections had marginal effects) were needed to induce anaboliceffects approaching those seen with IGF-I infusions.

Therefore, it was unclear whether the improved glycemic potency ofinjections of the pH5.4 formulation of IGF-I also would be translatedinto an improved anabolic efficacy of injections of IGF-I. In thisexample, hGH and IGF-I were co-injected twice daily to determine theresults.

Methods

1. Compounds

All solutions of IGF-I and GH alone or together were prepared on day 0so that sufficient drug was made for the entire example and stored at 4°C. during the seven-day experiment. The separate hGH and IGF-Iformulations employed were the pH 5.4 IGF-I formulation and the pH 6.0hGH formulation described in Example XIII.

2. Animals

Sixty female dw/dw rats of 60-70 days of age were obtained from Simonsen(Gilroy, CA). They were group housed in a room with controlled lightingand temperature and fed a grain diet and water ad lithium. They wereweighed twice and the largest and the smallest animals were discarded toleave 48 rats ranging in body weight from 105 to 135 grams at theirfirst injection.

3. Experimental Deigns

The rats were randomly assigned to six groups of eight rats per groupbased on their body weight, so that the average weight per group (119 g)was not different.

The six treatment groups were:

1) two excipient injections/day

2) pH 5.4 IGF-I formulation alone, 300 μg twice daily=600 μg/day

3) pH 6.0 hGH formulation alone, 15 μg twice daily=30 μg/day

4) pH 6.0 hGH formulation alone, 60 μg twice daily=120 μg/day

5) co-mix of pH 5.4 IGF-I formulation, 300 μg, plus pH 6.0 hGHformulation, 15 μg twice daily

6) co-mix of pH 5.4 IGF-I formulation, 300 μg, plus pH 6.0 hGHformulation, 60 μg twice daily

100-μl injections of each formulation were given subcutaneously in thenape of the neck twice daily either at the time the animals were weighed(8-9AM) or in the late afternoon (4-5 PM).

The last injection was a PM injection, with all the animals beingsacrificed approximately 18 hours later. At sacrifice, a large bloodsample was taken, and the liver, heart, spleen, thymus, and kidneys weredissected and weighed. Serum was assayed for IGF-I after extraction withacid-ethanol and a subsequent conventional RIA for IGF-I.

All data shown are Mean±SD with eight rats per group. Treatment groupswere compared by ANOVA and then by Duncan's Multiple Range Test.

Results

1. Body Weight Gain

The dose of IGF-I (300 μg/injection) was chosen as a near maximallyeffective hypoglycemic dose of IGF-I (based on the earlier examples)when given in the pH 5.4 formulation. These earlier examples used onlyone dose, so it was uncertain if a second or third dose would be more orless effective at inducing hypoglycemia. However, it was assumed thatthe 300 μg/injection dose would be nearly maximal as a hypoglycemic dosewhen given repeatedly, and that it also would be nearly maximal as ananabolic dose. The doses of hGH were based on effective dosesestablished in other experiments of twice daily hGH dosing in the dw/dwrat.

In addition, the doses of hGH and of IGF-I were chosen to attempt toobtain growth responses similar to those found with daily hGH dosing inthe dw/dw rat in Example II. Finally, the doses were also chosen to givea broad range of the hGH to IGF-I weight ratio (1:5 to 1:20) in therange where the hGH and IGF-I in the co-mix were shown to be chemicallystable.

A comparison of the results in the present example with the results inExample II show that comparable effects of IGF-I and hGH were found withthe dosing regimes chosen.

The animals appeared to tolerate the repeated injections of IGF-I withno overt signs of hypoglycemia. The anabolic or growth-promotingactivity of hGH and IGF-I was gauged primarily by measuring the weightgain in the rats. FIG. 25 and Table 2 show the weight gains of the dw/dwrats over the seven-day study. The control excipient-injected ratsshowed a very small weight gain (3.4±2.5 g over the 7-day study). Inother words, their growth reflected the GH- and IGF-deficient stateexpected of dwarf rats.

Twice daily IGF-I injections induced a surprisingly large, andstatistically significant (p<0.001), amount of body weight gain(11.9±2.5 g). These data are very similar to the data in FIG. 14, where240 μg/day of rhGH-I was infused for seven days in dw/dw rats andresulted in a weight gain of 12.15±3.75 g.

The two doses of rhGH both induced significant (p<0.001) weight gain(18.5±2.9 and 29-6±4.4 g for the 30 and 120 μg/day doses, respectively).The weight gain in Example II in the dw/dw rat with 200 μg/day of hGHgiven once daily was 19.9±4.5 g, which was similar to the response to 30μg/day of hGH in the present example.

There was an additive effect of the co-mixed IGF-I and hGH treatments.Thus, the weight gains for the co-mixed formulations of hGH and IGF-Iwere 25.4±3.9 and 36.1±4.9 g, for 30 and 120 μg hGH/day groups,respectively, compared to the 18.5±2.9 and 29.6+4.4 g gains for thesedoses given alone. The comparison of hGH alone to hGH+IGF-I wasstatistically significant (p<0.01) for both hGH doses.

In Example II, the weight gain with the 200 μg/day dose of hGH injectedonce daily along with an IGF-I infusion was 28.4+6.0 g, again similar tothe response to the 30 μg/day dosing of the hGH+IGF-I formulation in thepresent example. These data therefore indicate that with equivalentgrowth responses induced by hGH injection (whether given once or twice aday) the co-administration of IGF-I gives an additive growth response,irrespective of whether the IGF-I is given by infusion or by injection.

                                      TABLE 2                                     __________________________________________________________________________    Dw/dw rats treated with IGF-I + hGH; twice daily injection                                 Serum                                                                   Wt Gain                                                                             IGF-I  Kidney Wt                                                                           Thymus Wt                                                                            Spleen Wt                                    Group  (g)   (ng/ml)                                                                              (% Bwt)                                                                             (% Bwt)                                                                              (% Bwt)                                      __________________________________________________________________________    Excipient                                                                            3.4 ± 2.5                                                                        129 ± 49                                                                          0.86 ± .10                                                                       0.16 ± .02                                                                        0.23 ± .02                                IGF-I  11.9 ± 2.5*                                                                      140 ± 42                                                                          0.83 ± .05                                                                       0.17 ± .03                                                                        0.25 ± .02                                (600 μg)                                                                   hGH    18.5 ± 2.9*                                                                      163 ± 47                                                                          0.81 ± .07                                                                       0.17 ± .04                                                                        0.25 ± .02                                (30 μg)                                                                    hGH    29.6 ± 4.4*                                                                       189 ± 50*                                                                        0.84 ± .10                                                                       0.18 ± .03                                                                        0.27 ± .03*                               (120 μg)                                                                   IGF-I  25.4 ± 3.9*                                                                      120 ± 29                                                                          0.77 ± .04                                                                       0.16 ± .04                                                                        0.27 ± .02*                               (600 μg) +                                                                 hGH                                                                           (30 μg)                                                                    IGF-I  36.1 ± 4.9*                                                                      152 ± 41                                                                          0.77 ± .05                                                                       0.17 ± .03                                                                        0.31 ±  .04*                              (600 μg) +                                                                 hGH                                                                           (120 μg)                                                                   __________________________________________________________________________     Wt = weight                                                                   Bwt = body weight                                                             * = statistically significant                                                 (n = 8, p < 0.05 vs Excipient)                                           

2. Serum IGF-I Concentrations

The serum samples obtained at sacrifice 18 hours after the lastinjection were extracted and IGF-I was measured (Table 2). There was asmall but statistically significant rise in IGF-I after the injection ofhGH at the high dose. However, there was no maintained rise in serumIGF-I after the injection of IGF-I or after the injection of the co-mix,since IGF-I when given by injection is quite rapidly cleared in the rat.Sampling at times closer to the time of injection would be expected toshow the higher blood IGF-I concentrations seen in the earlier examples.The greater efficacy of the co-mix therefore could not be directly tiedto a higher serum concentration of IGF-I.

3. Organ Growth

The data in Table 2 for the growth of the different body organs gavesurprising results. In animals infused with IGF-I clear overgrowth ofthe spleen, thymus, and kidney has been shown in many studies, inhypophysectomized, dwarf, and normal animals. For example, see the datain Examples I and II above. However, in the present example, injectionsof IGF-I at doses that induced additive anabolic effects with hGH onwhole body growth gave surprisingly little organ overgrowth. These dataare quite different from the data with IGF-I infusions in the earlierexamples.

For instance, with IGF-I infusions in dw/dw rats in Example II, therelative size of the kidney increased significantly as a percent of bodyweight from 0.79±0.05 in excipient-treated controls to 1.0±0.09 inIGF-I-treated rats. However, in the present example (Table 2) when IGF-Iwas given by injection, with or without hGH, there was a tendency forthe relative size of the kidney to show, in fact, a reduction from0.86±0.10 in excipient-treated controls to 0.83±0.05 in IGF-I-treatedrats.

In Example II the thymus showed a vigorous growth response to IGF-Iinfusions, increasing well out of proportion to the rest of the body itsrelative size as a percent of body weight from 0.14±0.04 inexcipient-treated controls to 0.18±0.09 in IGF-I-treated rats. However,in the present Example (Table 2) IGF-I injections had no significanteffect on relative thymus weight whether IGF-I was given alone or withhGH.

The relative size of the spleen did show a significant increase withIGF-I treatment, but only when given along with highdose hGH treatment.In comparison, in Example II the relative size of the spleen increaseddramatically as a percent of body weight from 0.23±0.01 inexcipient-treated controls to 0.47±0.12 in rats treated with IGF-Ialone. In the present example, when IGF-I injections were given alonethere was no significant effect on absolute or relative spleen size, yetthere was a clear effect of IGF-I increasing spleen size when the IGF-Iwas given along with the hGH. This evidence suggests that the IGF-Iinjections in some way synergized with the hGH to induce spleen growth.

This relative lack of an effect of the IGF-I injections on the growth ofthe different tissues, compared to the effect of IGF-I infusions, madethe additive whole body effect of hGH and IGF-I even more surprising.Without being limited to any one theory, it appears that injections andinfusions of IGF-I cause a different spectrum of growth responses indifferent tissues. All the tissues measured seemed to respond to theIGF-I injections with a growth response more like that of GH in thatthey grew in proportion to the increase in whole body size. In theearlier examples, it might have been reasoned that GH and IGF-I mightsynergize to cause a whole body anabolic response because they causedselective and differential organ growth. However, when the IGF-I isgiven by injection there is much less evidence of selective ordifferential organ growth. It was surprising that the overgrowth of someorgans was lost when IGF-I was injected, yet when hGH was added to theformulation to be injected, the additive whole body anabolic activitywas retained.

Conclusion

Injections of the pH 5.4 formulation of IGF-I induced a very significantwhole body growth response with little organ overgrowth. In addition,when the IGF-I was co-injected with GH there was an additive anabolic orgrowth-promoting effect very similar to that seen previously with IGF-Iinfusions.

What is claimed is:
 1. A growth-promoting composition comprising IGF-Iand growth hormone, each in an amount of 0.1 to 100 mg/ml, in apharmaceutically acceptable carrier at a pH of about 6 containing asurfactant, wherein the amounts of IGF-I and growth hormone in thecomposition are effective to promote growth of a mammal more than anequivalent dose of IGF-I or growth hormone alone, and wherein the weightratio of IGF-I to growth hormone in the composition ranges from 0.002:1to 240:1.
 2. The composition of claim 1 wherein the amounts of IGF-I andgrowth hormone in the composition are about 1 to 10 mg/ml each and theweight ratio of IGF-I to growth hormone in the composition ranges from0.048:1 to 30:1.
 3. The composition of claim 1 wherein the carrier is anacetic acid salt buffer comprising a sodium counterion.
 4. Thecomposition of claim 3 wherein the surfactant is a polysorbate orpoloxamer.
 5. The composition of claim 4 further comprising astabilizer.
 6. A method for enhancing growth of a mammal comprisingadministering to the mammal by injection an effective amount of thecomposition of claim 1 so as to enhance the growth of the mammal overthe enhancement in growth achieved using an equivalent dose of IGF-I orGH alone.
 7. A method for enhancing growth of a mammal comprisingadministering to the mammal by infusion an effective amount of thecomposition of claim 1 so as to enhance the growth of the mammal overthe enhancement in growth achieved using an equivalent dose of IGF-I orGH alone.
 8. A composition comprising IGF-I and growth hormone in aweight ratio of IGF-I:growth hormone of between about 2:1 and 100:1(w/w), about 0.05-0.3of an osmolyte, about 0.1-10 mg/ml of a stabilizer,about 1-5 mg/ml of a surfactant, and about 5-100 mM of a buffer at aboutpH 5-6.
 9. The composition of claim 8 wherein the osmolyte is aninorganic salt and the surfactant is nonionic.
 10. The composition ofclaim 9 wherein the inorganic salt sodium chloride or potassiumchloride, the stabilizer is phenol or benzyl alcohol, the surfactant ispolysorbate or poloxamer, the buffer is sodium acetate or sodium citrateor both, and the amounts of IGF-I and growth hormone are about 2-20mg/ml and about 0.2-10 mg/ml, respectively, with the weight ratio ofIGF-I:growth hormone being between about 2:1 and 50:1.
 11. Thecomposition of claim 10 wherein the amount of IGF- I is about 7-10mg/ml, the amount of growth hormone is about 0.2-1.5 mg/ml, the weightratio of IGF-I: growth hormone is about 3:1 to 20:1, the amount ofsodium chloride is about 5-7 mg/ml, the amount of phenol is about 0.1-3mg/ml or the amount of benzyl alcohol is about 6-10 mg/ml, thesurfactant is polysorbate in an amount of about 1-3 mg/ml, the amount ofsodium acetate is about 2.5-4 mg/ml, and the amount of sodium citrate isabout 0.1-1 mg/ml.
 12. The composition of claim 8 that is lyophilized.13. A method for enhancing growth of a mammal comprising administeringto the mammal by injection an effective amount of the composition ofclaim 8 so as to enhance the growth of the mammal over the enhancementin growth achieved using an equivalent dose of IGF-I or GH alone.
 14. Amethod for enhancing growth of a mammal comprising administering totheμl by infusion an effective amount of the composition of claim 8 soas to enhance the growth of the mammal over the enhancement in growthachieved using an equivalent dose of IGF-I or GH alone.
 15. Acomposition comprising IGF-I and growth hormone in a weight ratio ofIGF-I:growth hormone of between about 2:1 and 100:1 (w/w), about0.05-0.3 mM of an inorganic salt, about 0.1-10 mg/ml of a stabilizer,about 1-5mg/ml of a nonionic surfactant, and about 5-100 mM of a bufferat about pH
 16. A method for enhancing growth of a mammal comprisingadministering Go the mammal by injection an effective amount of thecomposition of claim 8, wherein the weight ratio of IGF-I:growth hormoneadministered is between about 2:1 and 100:1.